In 1947 Los Angeles, animated cartoon characters, or "toons", co-exist with humans, often employing their skills to entertain as film stars. Private detective Eddie Valiant, once a staunch ally of the toons alongside his brother and co-worker Teddy, has become a depressed alcoholic following Teddy's murder by an unknown toon five years earlier. Maroon Cartoon Studios owner, R.K. Maroon, upset about the recent poor performance of his toon star Roger Rabbit, hires Eddie to investigate rumors that Roger's glamorous toon wife Jessica is having an affair with Marvin Acme, owner of both the Acme Corporation and Toontown, the animated metropolis in which toons reside.

After watching Jessica perform at The Ink and Paint Club, Eddie secretly photographs her and Acme playing patty-cake. He shows the pictures to Roger, who becomes distraught and flees, refusing to believe Jessica was unfaithful. The next morning, Acme is found murdered and evidence at the scene implicates Roger as a suspect. Eddie meets Judge Doom, the sinister human judge of Toontown—having bribed the electorate to gain their votes—and his five weasel minions, the Toon Patrol. Doom confirms he plans to execute Roger using the "Dip", a chemical concoction of acetone, benzene, and turpentine which is capable of destroying the otherwise invulnerable toons.

Roger's toon co-star, Baby Herman, suggests to Eddie that Acme's missing will—which supposedly bequeaths Toontown to the toons—may have been the killer's true motive. Eddie returns to his office and finds Roger waiting. Roger insists he has been framed and Eddie reluctantly agrees to help after finding evidence of Acme's will; he hides Roger in a bar tended by his girlfriend Dolores. Jessica tells Eddie that Maroon threatened Roger's career unless she posed for the compromising photos. Meanwhile, Dolores's research uncovers that Cloverleaf Industries recently bought the city's Pacific Electric railway system and will purchase Toontown at midnight unless Acme's will is found. Doom and the Toon Patrol find Roger, but he and Eddie escape with help from Benny, a toon taxi cab. Sheltering in a local theater, Eddie sees a newsreel of Maroon selling his studio to Cloverleaf.

While Eddie goes to the studio to interrogate Maroon, Jessica abducts Roger. Maroon denies involvement in Acme's murder, admitting he intended to blackmail Acme into selling his company as otherwise Cloverleaf would not buy the studio. Maroon is assassinated and Eddie spots Jessica fleeing the scene. Assuming she is the assailant, he reluctantly follows her into Toontown, choosing to discard the last of his alcohol. After saving Eddie from being shot by Doom, Jessica reveals her actions were to ensure Roger's safety and it was Doom who killed Acme and Maroon. Acme gave his will to Jessica for safety but, when she examined it, the paper was blank.

Doom and the Toon Patrol capture Jessica and Eddie, bringing them to Acme's factory. Doom reveals he is the sole shareholder of Cloverleaf and plans to erase Toontown with a Dip-spraying machine so he can build a freeway in its place, and decommission the railway system to force people to use it. When Roger unsuccessfully attempts to save Jessica, the couple is tied onto a hook in front of the machine's sprayer. Eddie distracts the weasels by performing a comedic vaudeville act full of pratfalls, causing them to die of laughter before he kicks their leader into the Dip. Doom is flattened by a steamroller while fighting with Eddie. However, Doom survives, revealing he is actually a disguised toon and Teddy's murderer. Struggling against Doom's toon abilities, Eddie empties the machine's Dip supply, spraying and dissolving Doom to death. The machine crashes through the wall into Toontown, where it is destroyed by a train.

As police and toons gather at the scene, Eddie realizes that Acme's will was written on the blank paper in temporarily invisible ink, confirming the toons inherit Toontown. Having regained his sense of humor, Eddie happily enters Toontown alongside Dolores, Roger, Jessica, and the toons.
Cast
Bob Hoskins plays the role of Eddie Valiant.
Live-action cast

    Bob Hoskins as Eddie Valiant, a private investigator
    Christopher Lloyd as Judge Doom, a judge of the Toontown Superior Court
    Stubby Kaye as Marvin Acme, the owner of the Acme Corporation and Toontown
    Joanna Cassidy as Dolores, a bar waitress who is friends with Eddie
    Alan Tilvern as R.K. Maroon, the head of Maroon Cartoons
    Richard LeParmentier as Lt. Santino, a police lieutenant of the Los Angeles Police Department
    Richard Ridings as Angelo, a patron at the bar that Dolores works at and taunts Eddie's detective work
    Joel Silver as Raoul
    Paul Springer as Augie
    Mike Edmonds as Stretch
    Betsy Brantley as Jessica Rabbit's performance model
    Morgan Deare as an editor

Voice cast

    Charles Fleischer as:
        Roger Rabbit, a rabbit and cartoon short star
        Benny the Cab, a toon taxi cab
        Greasy, the second-in-command of the Toon Patrol
        Psycho, a member of the Toon Patrol
    Kathleen Turner (speaking voice) and Amy Irving (singing voice) as Jessica Rabbit (both uncredited), a human toon and Roger's wife
    Lou Hirsch as Baby Herman, a baby and Roger's co-star, who speaks normally when off set
    David Lander as Smart Ass, the leader of the Toon Patrol
    Fred Newman as Stupid, a member of the Toon Patrol
    June Foray as:
        Wheezy, a member of the Toon Patrol
        Lena Hyena, an unattractive toon human whom Eddie mistakes for Jessica
    Mel Blanc as:
        Bugs Bunny
        Daffy Duck
        Porky Pig
        Tweety
        Sylvester the Cat
    Joe Alaskey as Yosemite Sam
    Wayne Allwine as Mickey Mouse
    Tony Anselmo as Donald Duck
    Tony Pope as:
        Goofy
        Big Bad Wolf[11]
    Mae Questel as Betty Boop
    Russi Taylor as
        Minnie Mouse
        The Hummingbirds from Song of the South
    Pat Buttram, Jim Cummings (imitating Andy Devine) and Jim Gallant (imitating Walter Brennan) as Eddie's toon bullets which he once received from Yosemite Sam
    Les Perkins as Mr. Toad from The Adventures of Ichabod and Mr. Toad
    Mary T. Radford as Hyacinth Hippo from Fantasia
    Nancy Cartwright as a Toon shoe who gets dissolved in Dip
    Cherry Davis as Woody Woodpecker
    Morgan Deare as Bongo the Gorilla, the bouncer of The Ink and Paint Club
    Peter Westy as Pinocchio
    Frank Welker as Dumbo (uncredited)
    Richard Williams as Droopy
    April Winchell as Mrs. Herman and Baby Herman's "baby noises"
    Archival recordings of Frank Sinatra were used for the Singing Sword, whose character design is based on Sinatra.