- Spyder-Knight (Peter Parker) (First appearance)
- Ultimate Spider-Man (Miles Morales) (First appearance)
- The Goblin (Norman Osborn)
- Ultimate Goblin (Norman Osborn) (First appearance)
- Electro (Max Dillon)
- The Alchemist (Otto Octavius) (First appearance)
- J. Jonas James' Son (Spyder-Knight) (First appearance)
- Merlin (Spyder-Knight) (First appearance)
- J. Jonah Jameson (Ultimate) (First appearance) (As hologram)
- Spyder-Horse (Spyder-Knight) (First appearance)
- Ultimate Spider-Man (Peter Parker) (First appearance) (Grave)
- Earth-TRN458 (Spider-Knight)
- Earth-TRN457 (Miles Morales)
- Spyder-Knight is voiced by Christopher Daniel Barnes (Earth-12041 Electro's voice actor) who voiced Spider-Man in Spider-Man: The Animated Series.
- Miles Morales was voiced by Donald Glover. In May 2010, a fan suggested Glover for the role of Peter Parker in the then-upcoming The Amazing Spider-Man film, encouraging his supporters to retweet the hashtag "#donald4spiderman". The campaign, originally started to see how far social networking could carry a message, quickly gained a large following.The call for Glover to be allowed to audition for the role was supported by Spider-Man co-creator Stan Lee. Comics writer Brian Michael Bendis, who announced an African-American version of Spider-Man a year later, said he had conceived of the character before Glover's campaign went viral. Bendis gave credit to Glover for influencing the new hero's looks; on seeing him dressed as Spider-Man on Community (a nod to the campaign), Bendis said, "I saw him in the costume and thought, 'I would like to read that book.'"
- Spider-Knight is a medieval Spider-Man in an armour resembling Spider-Armor MK III.
- Earth-TRN457 Miles Morales' and Earth-TRN457 Goblin's "Ultimate" titles are obvious nods to the Ultimate Universe.