Remy (Patton Oswalt) lives in a rat colony in the attic of a French country home with his brother Emile (Peter Sohn) and his father Django (Brian Dennehy). Unlike his kin, Remy is a gourmet whose keen smell is put to use sniffing food sources for rat poison. But Remy dreams of finer pursuits, sneaking into the kitchen to read the cookbook of his hero: Parisian chef Auguste Gusteau (Brad Garrett), who appears to Remy in visions throughout the film to expand on his motto that "anyone can cook." Remy learns that Gusteau died after a harsh review from mean-spirited food critic Anton Ego (Peter O'Toole).
The rats all flee when the resident, an old woman, discovers the rat colony. Remy, separated from the others, floats to Paris in its sewers, following Gusteau's image to the chef's namesake restaurant, now run by former sous-chef Skinner (Ian Holm). As Remy watches from a kitchen skylight, Alfredo Linguini (Lou Romano), a young man with no culinary talent, arrives and is hired on at the request of his recently-passed mother to do janitorial duties. The boy, unknown to all but his mother, is in fact Gusteau's son. Linguini spills a pot of soup and attempts to cover up his mistake by adding nearby random ingredients. Horrified by Linguini's actions, Remy falls into the kitchen and though desperately trying to escape, cannot help but stop and attempt to fix the ruined soup. Remy is caught in the act by Linguini, who himself is caught by Skinner as he captures the rat, but not before some of the soup has been served. To everyone's surprise the soup is a success. The kitchen's sole woman cook, Colette (Janeane Garofalo), convinces Skinner not to fire Linguini provided he can recreate the soup. And thus begins an alliance, uneasy at first, by which Remy secretly directs Linguini. The two perfect a marionette-like arrangement by which Remy tugs at Linguini's hair to control his movements.
Skinner discovers that Linguini is Gusteau's son, which he hides to prevent Linguini from inheriting the restaurant, which would thwart his ambitions of exploiting Gusteau's image to market prepared frozen dinners. Suspicious of Linguini, Skinner plies him with fine wine in an unsuccessful attempt to discover the secret of his unexpected talents. The next morning, hung over and disheveled, Linguini nearly confides his secret to Colette. Desperately trying to stop Linguini, Remy pulls his hairs, making him fall on Colette, leading the two to kiss. They begin dating, leaving Remy feeling abandoned.
One night Remy and his colony are reunited. Remy argues with Emile and his father over his new career as a secret chef. In the process of scrounging food for the clan Remy discovers Gusteau's will which, after a chase by Skinner, he presents to Linguini. Linguini now owns the restaurant, fires Skinner, and becomes a rising star in the culinary world, attracting renewed interest from Anton Ego, who had written off the restaurant for dead. Linguini and Remy have a falling out, Linguini deciding he no longer needs Remy, and Remy retaliating to the snub by leading a kitchen raid for his rat colony.
Things come to a head the night of a planned review by Ego. Linguini, unable to cook without the rat's guidance, admits his ruse to the staff, leading them all to walk out. Colette returns after thinking through Gusteau's motto. Django, inspired by his son's courage, returns with the entire rat colony to cook under Remy's direction, while Linguini, discovering his true talent, waits tables on roller skates. Colette helps Remy prepare ratatouille, a dish so good that Ego, in an epiphany at the climax of the film, relives memories of his childhood after taking a bite. Ego asks to meet the chef, but Colette tells him he must wait until the rest of the diners have left. At the end of the service, Remy and the rats are revealed. A changed man, Ego declares in his review that the chef at Gusteau's is the greatest chef in all of France.
In the denouement the restaurant is closed forever by a health inspector, who finds the rats after being tipped off by Skinner. Ego loses his credibility and job when the public discovers he has praised a rat-infested restaurant. Everything is for the best, however. With investments and regular visits from Ego, Linguini, Colette, and Remy open a successful new bistro called "La Ratatouille," which includes a kitchen and dining facilities for both rats and humans.
