- Professor Charles Xavier (Apparent death)
- Iceman (Bobby Drake)
- Kitty Pryde (First full appearance) (Joins team)
- Storm (Ororo Munroe)
- Colossus (Peter Rasputin) (Joins team)
- Rogue (Marie) 
- Wolverine (Logan)
- Cyclops (Scott Summers) (Death)
- Beast (Dr. Hank McCoy) (First appearance historically) (Rejoins team) (Last appearance chronologically)
- Xavier School Students
- Brotherhood of Mutants (Disbands)
- Magneto (Erik Lehnsherr)
- Phoenix (Jean Grey) (Returns) (Joins team) (Death)
- Mystique (Raven Darkholme) (Impersonates Magneto) (Leaves team) (Last appearance chronologically)
- Pyro (John Allerdyce) (Last appearance)
- Multiple Man (Jamie Madrox) (Joins team) (Last appearance)
- Juggernaut (Cain Marko) (Only appearance) (Joins team)
- Omega Gang (Only appearance)
- Callisto (Only appearance; dies)
- Arclight (Only appearance; dies)
- Quill (Only appearance; dies)
- Psylocke (Only appearance) (Apparent death)
- Phat (Only appearance)
- Spike (Only appearance; dies)
- Glob Herman (Only appearance)
- Lizard Man (Only appearance)
- Ash (Only appearance)
- John Grey (Only appearance) (Only in flashback)
- Elaine Grey (Only appearance) (Only in flashback)
- Stan Lee (First appearance chronologically)
- Worthington Labs (Only appearance)
- Trask (Only appearance)
- United States Army
- Xavier School Students
- P. Xavier (Only appearance; dies) (Cameo)
- United Nations (Cameo)
- Dr. Moira MacTaggert (First appearance historically) (Last appearance chronologically)
Races and Species:
- Magneto's Helmet
- Cyclops' Visor
- The Cure (First appearance)
- Sentinel (First appearance historically; head only)
- Fastball Special
- X-Jet (Destruction)
A pharmaceutical company announces that it has developed an inoculation to suppress the X-gene that gives mutants their powers and makes them different from humans, offering the cure to any mutant who wants it. While some mutants are interested in the cure, including the X-Men's Rogue, many others are horrified by the announcement. In response to the news, the X-Men's adversary Magneto raises an army, warning his followers that the cure will be forcefully used to eradicate all mutant powers.
Cyclops, haunted by the memory of the dead Jean Grey, returns to the place where Jean had sacrificed herself to save the X-Men. Jean appears to Cyclops, and as the two kiss, Jean changes and appears to kill Cyclops. Sensing trouble, Professor Charles Xavier sends Wolverine and Storm to investigate. When they arrive, the two X-Men encounter telekinetically floating rocks, Cyclops' glasses, and an unconscious Jean.
Xavier explains that when Jean sacrificed herself, she unleashed the powerful alternate personality she calls "Phoenix". Wolverine is disgusted to learn that Xavier has kept Jean in check telepathically, but when Jean awakens, he realizes she is not the Jean Grey he knew. Jean pleads with Wolverine to kill her, but when he refuses, the Phoenix surfaces and telekinetically slams Wolverine into a wall. She then escapes to her childhood home.
Magneto, also aware that Jean is now a powerful mutant, meets Xavier at Jean's house. The two men vie for Jean's loyalty until the Phoenix resurfaces, unleashing her power. She destroys her family's house, disintegrates Xavier, and leaves with Magneto.
Following the loss of Xavier, the X-Men regroup and confront Magneto's army, which is attacking the pharmaceutical company. During the fight, Beast injects Magneto with the cure, nullifying his mutant powers. After the battle, the Phoenix emerges and begins to destroy everything around her. Momentarily gaining control, Jean begs Wolverine to save her. Telling Jean he loves her, Wolverine reluctantly kills her with his claws.
Despite the X-Men's losses, life goes on. Magneto, now an ordinary man, sits at a chessboard and reaches out toward a metal chess piece that trembles slightly, hinting that the cure may not be as permanent as thought. Following the end credits, Dr. Moira MacTaggert checks on a comatose patient, P. Xavier, Charles' brother. He calls out to her with Charles's voice. Startled, she replies, "Charles?"
- Hugh Jackman as Wolverine / Logan
- Halle Berry as Storm / Ororo Munroe
- Ian McKellen as Magneto / Erik Lehnsherr
- Famke Janssen as Phoenix / Jean Grey
- Haley Ramm portrays a young Jean Grey
- Anna Paquin as Rogue / Marie
- Kelsey Grammer as Beast / Dr. Henry "Hank" McCoy
- James Marsden as Cyclops / Scott Summers
- Rebecca Romijn as Mystique / Raven Darkholme
- Shawn Ashmore as Iceman / Bobby Drake
- Aaron Stanford as Pyro / John Allerdyce
- Vinnie Jones as Juggernaut / Cain Marko
- Patrick Stewart as Professor Charles Xavier
- Elliot Page as Kitty Pryde
- Ben Foster as Angel / Warren Worthington III
- Cayden Boyd portrays a young Warren Worthington III
- Dania Ramirez as Callisto
- Michael Murphy as Warren Worthington II
- Shohreh Aghdashloo as Dr. Kavita Rao
- Josef Sommer as the President of the United States
- Bill Duke as Bolivar Trask
- Daniel Cudmore as Colossus / Peter Rasputin
- Eric Dane as Multiple Man
Additionally, Adrian Hough and Desiree Zurowski portray Mr. Grey and Mrs. Grey, respectively. Chris Claremont and Stan Lee make cameo appearances. Cameron Bright portrays Leech / Jimmy. Connor Widdows, Kea Wong, Shauna Kain, and Luke Phol appear as Xavier's School for Gifted Youngsters students Jones, Jubilee / Jubilation Lee, Siryn / Theresa Cassidy, and Flea, respectively. Lloyd Adams, Meiling Melançon, Omahyra Mota, Clayton Watmough, and Ken Leung portray Omegas Lizard Man, Psylocke, Arclight, Glob Herman, and Kid Omega, respectively. Phat is portrayed by Via Saleuma and Richard Yee. R. Lee Ermey makes an appearance as a sergeant on Alcatraz Island. Lance Gibson appears as Spike. Olivia Williams makes an uncredited appearance as Dr. Moira MacTaggert.
- In July 2004, Bryan Singer, the director of the first two X-Men films, left the project during early pre-production, in order to direct the film Superman Returns. He was joined by X2 screenwriters Dan Harris and Michael Dougherty, composer/editor John Ottman, and cast member James Marsden. Among the choices for replacements were Darren Aronofsky, Joss Whedon, Rob Bowman, Alex Proyas, and Zack Snyder.
- In March 2005, Matthew Vaughn was hired as the new director for the project. He cast Kelsey Grammer as Beast / Dr. Henry "Hank" McCoy, Dania Ramirez as Callisto and Vinnie Jones as Juggernaut / Cain Marko, but family issues reportedly led him to withdraw before shooting began. Vaughn was replaced by Singer's friend Brett Ratner, who was among those originally considered to direct the first film — and coincidentally was considered by Warner Brothers to direct the 2006 Superman project before it evolved into Superman Returns.
- The film was originally scheduled for a May 5, 2006 release, but was pushed back to May 26 of the same year due to production delays.
- The film began filming in August 2005 and ended in January 2006.
- Many of the cast members from the previous two X-Men films returned, the major exceptions being Alan Cumming, who portrayed Nightcrawler / Kurt Wagner and Kelly Hu, who portrayed Deathstrike / Yuriko Oyama, from X2. Both of whom signed two-film contracts. However, Hu's character was killed by Wolverine in the previous film and Alan Cumming was reportedly uncomfortable with the long hours he had to take with the Nightcrawler makeup. The part for Nightcrawler was so minimal in the third film that he felt it was not worth it to go through the long and costly makeup process when he was barely in the film, and the character was cut. He did agree, however, to do voice work for the character for the video game based on the film. James Marsden did appear in the film as Cyclops / Scott Summers, despite reported scheduling conflicts with Superman Returns.
- Halle Berry stated during interviews for X2 that she would not return as Storm in the third film unless the character had a significant presence comparable to the comic-book version. Brett Ratner also felt Storm required a larger role and there was little difficulty reaching an agreement.
- Filmmakers considered using the Beast character since the first X-Men movie, but budget constraints ruled him out; however, Steve Bacic is identified as Hank McCoy when he appears on a television screen in a cameo role in X2.
- The events of this movie occur in 2006.
The novelization of the film, written by comic book writer Chris Claremont, was released on May 16, 2006. The novelization of the movie differs significantly from the film.