Superman finds himself trapped in a topsy-turvy world, where the Justice League of America have become villainous mockeries of themselves (he himself being a criminal), while the "real" Justice League is made up of various supervillains, and led by Bizarro. As Superman continues to try to make sense out of such a nonsensical, insane world, he slowly comes to the realization that it was not always like this.
- Justice League:
- Superman (George Newbern) - finds out from Mxyzptlk that Joker stole his powers
- Batman (Kevin Conroy) - killed several times by Joker to humiliate him
- Wonder Woman (Susan Eisenberg) - in Joker's world, she serves as a champion to women's right
- The Flash (Michael Rosenbaum) - in Joker's world, he is the Flub, the fastest fattest man alive
- Green Lantern (Phil LaMarr) - in Joker's world, he is Yellow Paper Lantern who can make shadow puppets
- Martian Manhunter (Carl Lumbly) - in Joker's world, he is a dwarf who longs to destroy Earth
- Hawkgirl (Maria Canals Barrera) - in Joker's world, she is a natural born Hawk as Joker's eyes.
- Kara In-Ze/Supergirl (Nicholle Tom) - in Joker's world, she lives in an orphanage run by Nun Perry White, she discovered visions of Superman's cry for help, and leaves the orphanage
- Barbara Gordon/Batgirl (Tara Strong) - in Joker's world, she dressed as a mermaid in Joker's Laughing Fish commercial
- Dick Grayson/Nightwing (Loren Lester) - in Joker's world, works as a register guy at Joker's Laughing Snack Time restaurant
- Tim Drake/Robin (Eli Marienthal) - in Joker's world, he dressed as a chicken mascot at Joker's Laughing Snack Time
- Bizarro (George Newbern) - at the beginning introduced as the main antagonist, only to be revealed as pawn for promotional campaign to cover Joker's status
- Emperor Joker (Mark Hamill) - the main antagonist, having stolen Mr. Mxyzptlk's powers, he remade the world in his own image, and kills and tortures Batman several times
- Empress Harley Quinn (Tara Strong) - Joker's empress, who winches in terror when he kills Batman in horrific ways, when she tried to convince him not to go inside Batman's brain, he shuts her vocal cords, rendering her muteless
- Lex Luthor (Clancy Brown) - the secondary antagonist, on January 2017, he was going to be president of the United States, only to be outranked by Joker and put into a jester's costume by him after he gained Mxyzptlk's powers, and vows to get even with him.
- Dr. Polaris (Sean Donnelon) - the tertiary antagonist, serves as Joker's advisor in his kingdom.
- Amanda Waller (C.C.H. Pounder) - the quaternary antagonist, is seen as the Hippopotamus at the theme park entrance.
- Affected by Joker's new powers:
- Killer Croc - as a head knight of Joker's orders, shown to be more Crocodile than half.
- Two-Face - with features of Comedy and Tragidy on his face.
- Penguin - serves as thr Joker's man servant.
- Scarecrow - No longer fear but silliness.
- Riddler - serves as the Game show host.
- Poison Ivy - is a humanoid Tree that acts as Joker's guard.
- Catwoman - Black and White bikini wearing Catwoman who serves as Joker's amusement, (much to Harley's jealousy)
- Wildcat - serves as the guard of the door.
- Creeper - serves as the pet of Joker.
- Dr. Fate - is the main wizard for Joker.
- Alfred Pennyworth - is a prison guard.
- Emil Hamilton - serves as Joker's adapter.
- Lois Lane (Diane DeLano) - bald and evil, thanks to Joker's machinations
- Jimmy Olsen (David Kaufman) - in Joker's world, begging for peace dressed like the classic Superman.
- Commissioner Jim Gordon (Darin DePaul) - removed from his uniform into his boxers and tank top by Joker during the song, "Where's the Fun in That?"
- Harvey Bullock (Vincent D. Onifrio) - removed from his uniform into his boxers and tank top by Joker during the song, "Where's the Fun in That?"
- King Faraday (Scott Patterson) - trying to escape Joker's horrible maze.
- Emil Dorian (Xander Berkley) - a scientist for Joker.
- The film is rated PG-13 for ages 13 and up.
- The Character designer is Bruce Timm.
- Differences from the comic:
- Joker sings the song, "Where's the Fun in That?" from the episode of Batman: The Brave and the Bold", only to jazz it up a bit instead of rock n roll, he does it in Hip-Hop.
- Kara In-Ze replaces Matrix Supergirl, while Nightwing, Batgirl and Robin replace Superboy
- John Stewart is the Yellow Paper Lantern, and Hawkgirl replaces both Plastic Man and Aquaman