- Captain America (Steve Rogers) (Main story and flashback)
- Falcon (Samuel Wilson) (First appearance)
- S.H.I.E.L.D. (Disbands)
- Black Widow (Agent Natasha Romanoff/Natalia Romanoff) (Leaves team) (Real name first revealed) (Impersonates Councilwoman Hawley)
- Nick Fury (Leaves team)
- Agent Maria Hill (Leaves team)
- Agent 13 (Sharon Carter) (First appearance) (Leaves team)
- Agent Cameron Klein (First appearance)
- Agent Moore (First appearance)
- Peggy Carter
- World Security Council
- S.T.R.I.K.E. (Disbands)
- Agent Jasper Sitwell (Death)
- Secretary Alexander Pierce (Only appearance; dies)
- Winter Soldier (Bucky Barnes) (Main story and flashback) (First appearance as Winter Soldier) (Revealed to be alive) (Leaves team)
- Arnim Zola (Death) (Main story and flashback)
- Senator Stern
- Project Insight (First appearance)
- Agent Russo (First appearance)
- Baron Wolfgang von Strucker (First appearance) (Cameo)
- Doctor List (First appearance) (Unnamed) (Cameo)
- Pietro Maximoff (First appearance) (Unnamed) (Cameo)
- Wanda Maximoff (First appearance) (Unnamed) (Cameo)
- Georges Batroc's Mercenary Group (First appearance)
- President Matthew Ellis (Photo) (Full name revealed)
- Howling Commandos (On-screen)
- Garcia (First appearance)
- Metropolitan Police Department of the District of Columbia (First appearance)
- Aaron (First appearance)
- SSR (On-screen)
- Red Skull (Johann Schmidt) (On-screen)
- Operation Paperclip (On-screen)
- Obadiah Stane (Named only)
- Renata (Only appearance; dies)
- KGB (Mentioned)
- Riley (Photo)
- Bruce Banner (Mentioned)
- Stephen Strange (Mentioned)
- Dr. Fine (First appearance)
- Sarah Rogers (Mentioned)
- Stan Lee
- Lt. Marcus Bledsoe (First appearance) (Appears on a computer screen, TV, or hologram only)
- Michael Lindon (First appearance) (Appears on a computer screen, TV, or hologram only)
- C.I.A. (First appearance)
- Stark Industries (Cameo)
- Iron Man (Tony Stark) (Photo)
- United States Department of Defense (First appearance)
Races and Species:
- Humans (Main story and flashback)
- Earth-199999 (Main story and flashback)
- Earth (Main story and flashback)
- North America (Main story and flashback)
- United States of America (Main story and flashback)
- Washington, D.C.
- Maryland (On-screen)
- Ellicott City (On-screen)
- New Jersey (Main story and flashback)
- Wheaton (Main story and flashback)
- Camp Lehigh (Main story and flashback)
- Wheaton (Main story and flashback)
- New York (Main story and flashback)
- Iowa City, Iowa (Mentioned)
- United States of America (Main story and flashback)
- Indian Ocean
- Algeria, Africa (Mentioned)
- Algiers (Mentioned)
- Cairo, Egypt, Africa (Mentioned)
- North America (Main story and flashback)
- Earth (Main story and flashback)
- Super-Soldier Serum (Mentioned)
- Captain America's Shield
- Captain America's Uniform
- Black Widow's Gauntlets
- Black Widow's Suit
- Mousehole (First appearance)
- Taser Rod (First appearance)
- Winter Soldier's Bionic Arm (First appearance)
- Arnim Zola's Computer Body (First full appearance) (Destruction)
- Project Insight satellites
- Exo-7 Falcon (First appearance)
- Memory Suppression Device (First appearance)
- Photostatic Veil (First appearance)
- Loki's Sceptre (Cameo)
- Mind Stone (Behind the scenes) (Cameo)
- Milkor Grenade Launcher
- Chevrolet Corvette Stingray C7
- S.H.I.E.L.D. Quinjets
- Lemurian Star
- Captain America's Motorcycle (Harley-Davidson)
- S.H.I.E.L.D. Flying Car
- S.H.I.E.L.D. Helicarriers
- Chevrolet Impala 9C1 police cruiser
Two years after the Battle for New York, Steve Rogers, a.k.a. Captain America, is jogging in Washington, D.C., and frequently passes by a man who later introduces himself as Samuel Wilson, an army veteran who now works in the debriefing section. After the jog, Sam and Steve have a chat about how Steve is adapting to the new world. Natasha Romanoff then arrives to take Steve with her on a mission. The mission at hand is to rescue the hostages of a hijacked S.H.I.E.L.D ship. Cap, Agent Rumlow, Romanoff and his men fight the pirates and rescue the hostages. But as it turns out, Natasha had already gotten a secret mission which was to retrieve some S.H.I.E.L.D data from the ship to a USB stick. Steve was annoyed that he had not been privy to all the details of the mission.
Back at the Triskelion, Steve confronts Nick Fury about Black Widow's secret mission. Fury then takes him to the lower levels of the building where three Helicarriers are being prepared, introducing Rogers to the Project Insight. He tells Steve that S.H.I.E.L.D treats the world as it is, not as they would like it to be, and that they are going to use the Helicarriers to fight threats before they even happen. Steve states that "This isn't freedom. This is fear."
Later, Steve visits the Captain America exposition at the Smithsonian Institution to remember the past, afterward, he visits Peggy Carter in a retirement home, where she is now past 90 years old. He later meets with Samuel Wilson again after watching him during the end of his debriefing session. Meanwhile, Fury discovers that the data on the USB extracted by Widow was inaccessible. He tells this to Alexander Pierce, who just had a talk with the World Security Council about whether or not to let Fury continue to run S.H.I.E.L.D.. Later, Fury is on his way home from the Triskelion when he is attacked by false cops. He defeats them but is stopped when a masked man shoots a magnetic bomb under Fury's car, flipping it over. But before the attacker gets to Fury, he manages to drill a hole in the ground using a blowtorch.
Steve arrives to his apartment after talking to his neighbor Kate, but gets suspicious when he hears his stereo's still on. He sneaks in through the window and finds Nick Fury sitting in his couch. Fury reveals S.H.I.E.L.D. has been compromised and that they might be overheard by someone, before getting shot several times in his back through the wall. He gives the USB to Steve and tells him not to trust anyone, before falling unconscious. Roger's neighbor smashes in the door revealing herself to be an undercover S.H.I.E.L.D. agent assigned to protect Rogers, Agent 13. Cap finds the gunman and starts to chase him. He throws the shield at him, but surprisingly the man catches the shield with his cybernetic arm and throws it back. He then jumps of the building and disappears.
Fury is taken to a hospital but his heart stops and he couldn't get revived. Apparently dead, he's taken away and Agent Maria Hill asks to take care of the body. Afterwards, Agent Rumlow arrives to tell Steve that he is wanted for a hearing. He quickly hides the USB in a vendor machine and leaves. When he arrives at the Triskelion, he is questioned by Alexander Pierce who says he is one of Nick's oldest friends and wants to know who killed him. He also states that he and Nick were realists and that to build a new world sometimes meant tearing the old one down. Cap doesn't trust Pierce and tells him exclusively that Fury told him not to trust anyone. He then leaves Alexander to get back to the hospital. When in the elevator, it starts to fill up with people and Steve soon realizes that they are there to take him down. He asks if anyone wants to get out and then the fight begins. It's hard but Cap manages to knock out all his attackers, before jumping out the elevator to avoid arrest. After surviving the fall he drives away on a motorcycle. A roadblock is set up for him and also a Quinjet attempts to stop him. He does however get past both.
It is later revealed that Pierce is working with the masked man, and Alexander gives him a new mission.
Steve later arrives to the hospital where he notices that the USB is gone from the vending machine. Natasha had taken it and he angrily asks her what she knows about what has happened. She tells him that the masked man is an assassin known as the Winter Soldier, who she had encountered once before. She decides to follow Cap. They go to an Apple store and use one of the computer to see if there is any valuable information on the flash drive. But Natasha fails to open the files. However, she manages to find the location where the USB could be unlocked. Rumlow and his team track them to the mall and try to find them, but fail.
Steve and Natasha steal a car and drive to the location, New Jersey, while talking about the past. When they arrive Steve realizes that they are at Camp Lehigh, the Army base where he trained during WWII. After a moment of nostalgia, Steve realized that one of the buildings - an ammo dump - was not there seventy years ago, and should not be there, as Army regulations forbid the storage of weapons within 500 feet of a barracks for safety reasons. Inside the "ammo dump", they find not munitions but a first-generation S.H.I.E.L.D. office - and a secret elevator leading to a room filled with old computers... fitted with a modern USB interface.
Fury's USB drive causes the computers to power up, and someone starts to talk to them. It is revealed to be Arnim Zola, who tells them that he was recruited by S.H.I.E.L.D. after the war and that he helped to rebuild Hydra inside the organization. In the 1970s, he received the news that he had a terminal condition, and, S.H.I.E.L.D, still needing his help, transferred his mind into a software. He also had been working on an algorithm that Hydra wanted which allowed to determine someone's future by their past records. He tolled them that some of the most important assassinations through history had been due to Hydra, showing them a picture of Howard Stark as an example, implying that his death wasn't an accident. Zola also reveals he was involved in the Winter Soldier program. But before Rogers and Romanoff can get any more information out of Zola, a missile launched by S.H.I.E.L.D. blows up the base. They survive the explosion and seek refuge with Sam Wilson. Sam tells them that he was part of a military project called Falcon, a testing of a new form of winged jetpacks.
After retrieving Sam's former Falcon jetpack from custody, Steve, Natasha and Sam capture S.H.I.E.L.D. agent Sitwell, who has been working for Hydra. He just had a meeting with Senator Stern, who before saying goodbye whispered "Hail Hydra." Sam Wilson calls Sitwell and forces him up to the roof of a tall building together with Natasha and Steve they get him to tell them that Zola's algorithm could calculate if a person would be a threat for Hydra's plans. He also revealed that Hydra is going to use Project Insight to eliminate the millions of people stated to be threats for their plans, such as Tony Stark, Bruce Banner and Stephen Strange.
The heroes leave but they are attacked by the Winter Soldier and his men. A battle breaks out, and, as Cap and the Winter Soldier fight, his mask is broken and his identity is revealed, Bucky Barnes, Steve's old friend thought dead during the WWII. Cap tries to see if Bucky recognises him, but he doesn't show any signal of doing so. Steve, Natasha and Sam are taken into custody by S.H.I.E.L.D.. While in the prison van, one of the guards stuns the other one and reveals herself to be Maria Hill. She then uses the blowtorch to escape with them.
Agent Hill takes them to a secret facility where Nick Fury is resting, revealing that he didn't die, even though he is severely injured. He had been injected an anti-stress serum at the hospital, which had been developed by Bruce Banner, that lowered his heartbeat to only one beat per minute so that he would appear dead. Hill and Fury inform Steve that Project Insight was only hours away and that they have to act quickly. The Helicarriers would hover in a triangle, connected, targeting all its targets before firing. The only way to prevent this was to use three special chips that would take away S.H.I.E.L.D.'s control over the Helicarriers. Cap also decides to steal a replica of his WWII costume from the Captain America exposition, as he needed an uniform.
The Winter Soldier is being prepared for battle but he starts to ask who Bucky is. Hydra decides he is remembering too much about his past and they brainwash him again. He remembers how Hydra's Russian division found him after falling off the train, that Zola had replaced his left arm for a cybernetic one, and that we was placed in cryo-tanks between his missions so that he would not age.
As the members of the World Security Council arrive at the Triskelion, they are given a device to be put on their collar that will grant them access to anywhere in the Triskelion. They then all walk up to Alexander Pierce's office. Captain America and Falcon break in and take control of the loudspeakers. Steve proceeds to reveal that S.H.I.E.L.D. was under the influence of Hydra, whose leader was Alexander Pierce, and that innocent people would die if Project Insight was activated. Rumlow and his men walk into the control room and threaten to shoot the agent in charge if he doesn't get the Helicarriers in the air. Agent 13 attempts to intervene, but fails and Rumlow still manages to get the Helicariers in the air.
Captain America and Falcon manage to change the chips of two of the Helicarriers, but as he they were approaching to the third one, the Winter Soldier damages Falcon's jetpack, forcing Sam to open a parachute and get down to the Triskelion, where he gets caught up in a fight against Rumlow. Captain America, however, manages to get to the panel where the chip must be changed, but he's confronted by Bucky. Against his will, Cap fights his former friend as he tries to make him remember who he was. After knocking out Bucky, a heavily injured Cap manages to get the chip in place and allow Hill to take control of the Helicarriers and make them fire at each other.
As his true intentions were revealed, Pierce takes the World Security Council hostage. But Black Widow, who was revealed to have impersonated one of the members stops him. She then calls for Fury to force Pierce unleash the secrets of S.H.I.E.L.D. and Hydra to the world, as two agents of the highest levels are required to do so. As the files are being uploaded, Pierce activates the devices in the collars of the rest of the Security Council, which kills them. Pierce attempts to use Natasha as a gateway, but she uses a micro-EMP to deactivate the device, giving Fury the freedom to shoot Pierce, who dies.
One of the Helicarriers crashes into the Triskelion and causes it to collapse. Both Falcon and Rumlow run from the debris, but the latter is berried under rumble as Falcon jumps out of a window and is saved by Fury and Black Widow in a helicopter.
As the Helicarrier they were aboard starts collapsing, Steve finds Bucky trapped under rubble. Cap rescues him, but the Winter Soldier continues with his mission and attacks him. Refusing to fight who once was his best friend, Steve throws his shield of the Helicarrier and lets the Winter Soldier beat him. But Bucky snaps out and stops attacking Steve. Cap then falls into the water of the Potomac River and is about to drown when the Winter Soldier pulls him to the side of the river before disappearing.
Later, Steve wakes up in a hospital with Sam accompanying him. Rumlow is found alive and is attended by doctors. Agent 13 is now a member of the CIA. Maria Hill is seen applying for a job at Stark Industries. Fury burns down all his possessions. Natasha appears before a Senate subcommittee. Steve, Sam and Fury gather in Fury's grave as the latter informs Steve he will go undercover to Europe to hunt down Hydra cells. As Fury leaves the scene, Natasha appears to give Steve a dossier on Bucky and the Winter Soldier program she recovered from Kiev. Steve wants to track down Bucky, and Sam decides to join him.
In a mid-credits scene, Hydra agent Baron Wolfgang von Strucker discusses with his subordinates how even though the Hydra agents in S.H.I.E.L.D. were gone there are still followers all around the world. Strucker also revises their experiment on the Chitauri spear taken from Loki after the Battle for New York. He then walks over to a pair of cells, talking about some of his failed experiments. He stops in front of two cells, and talks about two of his test subjects who survived, referring to them as "the twins". One of the cells contains a man who is running around at a tremendous speed. The other one contains a woman who levitates a few blocks, before crushing two blocks together. Von Strucker tells one of his men, "It's not a world of spies anymore, Doctor. Not even a world of heroes. No... this... is the age of miracles... And nothing is more horrifying than a miracle."
In a post-credits scene, Bucky, now in normal clothes, is touring the Captain America exhibit at the Smithsonian Institution when he finds with a section dedicated to himself.
Additionally, Garry Shandling reprises his role as Senator Stern. Adetokumboh M'Cormack portrays Ferdinand Lopez. Aaron Himelstein appears as a launch technician. Director Joe Russo makes a cameo as Dr. Fine. Writers Christopher Markus and Stephen McFeely make cameos as SHIELD interrogators. Ed Brubaker, DC Pierson, Danny Pudi, and Stan Lee make cameos in the film. Branka Katić portrays Renata. Steven Culp appears as Congressman Wenham, although he is credited as Scudder. Nestor Serrano portrays General Scudder. Gary Sinise voiced the narrator of the Smithsonian's Captain America exhibit. In the mid-credits scene, Aaron Taylor-Johnson, Elizabeth Olsen, Henry Goodman and Thomas Kretschmann make uncredited appearances as Pietro Maximoff, Wanda Maximoff, Doctor List and Baron Wolfgang von Strucker, respectively.
- Joss Whedon directed the post-credits scene featuring Baron Strucker, Wanda Maximoff, and Peter Maximoff, who next appeared in Avengers: Age of Ultron, which he also directed.
- The mural of Captain America and the Howling Commandos seen in the Smithsonian Institution was painted by Marvel Studios' Head of Visual Development Ryan Meinerding, who also designed the Iron Man Armor, some of them along with Adi Granov.
- The home video release for Captain America: The Winter Soldier will be the first to not include a Marvel One-Shot since Marvel began including of the short films with the home video release of each film in the Marvel Cinematic Universe. The last home video release not to be paired with a One-Shot was Iron Man 2.
- Hawkeye appeared in early drafts of the film. He would've been featured in an scene set between Captain America's escape from the Triskelion and his arrival to the hospital to retrieve the USB stick. Hawkeye would have confronted Captain America in the outskirts of Washington, D.C., with both of them closing in on each other. After Cap took down Hawkeye, he would have realized Clint was trying to trick S.H.I.E.L.D., Clint would've alerted Rogers that his uniform had a tracking device, and would've asked Steve to punch him to make the fight look real, as there was a Quinjet hovering above where they were watching the feedback back at S.H.I.E.L.D.. This scene would've been shot if there hadn't been a conflict with Jeremy Renner's schedule.
- The five first subjects in the list from the page shown of Steve Rogers' notebook in which he lists numerous things he missed while being frozen differ from region to region. The American version includes "Steve Jobs" and "Disco," the British version includes "The Beatles" and "Sean Connery," the French version includes "Daft Punk" and "France '98," the Italian version includes "Roberto Benigni" and "Ferrari's victories at F1 Grand Prix," the Spanish version includes "Rafa Nadal" and "1978 Constitution," the Latin American version includes "Maradona's Hand of God" and "Shakira," the Russian version includes "Yuri Gagarin" and "Soviet Union Dissolution - 1991," the South Korean version includes "Ji-Sung Park" and "2002 World Cup," and the Australian version includes "AC/DC" and "Skippy, the Bush Kangaroo." The subjects "Thai food," "Star Wars / Trek," "Nirvana (Band)," "Rocky (Rocky II?)," and "Troubleman (Soundtrack)" are included in every version. The American and British version share "Moon Landing" and the American and Italian version share "Berlin Wall (up + down)."
- The scene in which Captain America jumps off the Quinjet without parachute is a homage to a similar scene in Ultimates 2 #1.
- Steve and Bucky's first exchange of the film is a direct quote of Captain America #8.
- When Black Widow boots up Arnim Zola's program, she says, "Shall we play a game?" This is a reference to a line asked by the computer in the film WarGames.
- Nick Fury's headstone has on it a quote from Ezekiel 25:17... "The path, of the righteous man..." This is a reference to Samuel L Jacskson's part in the movie Pulp Fiction.
- HYDRA's infiltration of S.H.I.E.L.D was an important storyline in the comic book series Secret Warriors Vol 1.