The film opens with a television documentary detailing the life of Auguste Gusteau, a French chef who is the owner of a 5-star restaurant in Paris, the youngest chef to ever have a 5-star rating and the author of a best selling cookbook, Anyone Can Cook. His mantra of "Anyone can cook" is disbelieved by a ruthless food critic named Anton Ego.
The film then follows Remy, a rat living with his colony in the attic of an old woman's house in the countryside. His brother is named Emile and their father Django is the leader of the colony. Remy has a keen sense of taste and smell, which causes his father to give him a job smelling for poison in the food the rats steal. Remy despises eating garbage and is fascinated by humans and their ability to cook and create. Django forbids Remy to go into the kitchen (where all the food isn't poison) and away from the humans. But Remy ignores the warning and goes into the kitchen of the house and watches the documentary on Gusteau, who becomes his idol.
The next day, Remy cooks a mushroom with some cheese Emile found, and takes a huge liking to it. Remy wants to add saffron to it, so he and Emile go inside the kitchen. As Remy searches for it, he reveals that he can read. Remy finds out Gusteau has passed away after Ego's negative review cost the restaurant its 5-star rating. The old lady wakes up and is shocked to see both Remy and Emile. She grabs a shotgun and shoots at the two as they escape into the ceiling. The ceiling breaks open, revealing the entire rat colony, shocking the woman even more. The rats evacuate, but Remy stays behind to take the woman' cookbook. He tries to catch up with the colony, using the book as a raft and a spatula as a paddle, but takes a wrong turn and ends up separated from the colony.
Remy reads the cookbook and gets hungry. An imaginary figure of Gusteau tells him to search around. Remy makes his way up the sewers into an apartment complex, where he finds bread. Gusteau stops him from stealing the bread, telling him that "a cook makes, a thief takes." Remy heads to the roof of the apartment to find he's been in Paris this whole time, and sees Gusteau's restaurant across the street.
In the kitchen of the restaurant, a young man named Alfredo Linguini waits in to meet the head chef, Skinner, the restaurant's current owner and Gusteau's former sous-chef. Linguini is the son of Renata, one of Gusteau's ex-girlfriends who recently passed away. Linguini gives Skinner a letter from his mother in hopes that it'll help him get a job at the restaurant. Skinner doesn't read the letter, but gives Linguini a job as a garbage boy.
Meanwhile, Remy heads to the restaurant's kitchen skylight to watch the staff in action and identifies the roles of each kitchen staff. He watches in horror as Linguini spills some soup and attempts to recreate it using random ingredients. Enraged, Remy accidentally falls into the kitchen. As he is about to escape, he decides to fix the soup only to be caught by Linguini. Linguini traps Remy in a colander just as Skinner arrives to take the soup, who assumes Linguini is the one to alter the soup. Skinner fires Linguini, but when the customer, who is a food critic, praises the soup, the restaurant's only female chef named Colette convinces Skinner to retain Linguini. Skinner decides to keep Linguini and let Colette take care of him. As Remy is escaping, he is spotted by Skinner. Linguini traps Remy in a jar, and Skinner tells him to get rid of it away from the restaurant so that it doesn't get out the restaurant has vermin.
Linguini takes Remy to a river to drown him, but upon realizing that the rat can understand him, asks Remy to help him recreate the soup to impress Skinner. The next day, the two have a hard time overcoming their language barrier, until Remy starts pulling Linguini's hair to control his limbs like a marionette. With Remy controlling Linguini while hidden inside his toque, the pair successfully meet the challenges devised by Skinner. Colette begrudgingly trains Linguini, but steadily comes to appreciate someone heeding her advice. Remy finds Emile eating garbage outside the restaurant and reunites with the rat colony. When Remy admits he's been spending time with humans, Django shows him a rat extermination store to convince him humans are dangerous, but Remy ignores his warnings and leaves.
Skinner reads Renata's letter, which reveals Linguini is Gusteau's son and the rightful owner of the restaurant. Skinner is shocked and enraged about this revelation, as it would ruin Skinner's plans to use Gusteau's name to market a line of microwaveable meals. Gusteau's will stated that Skinner would inherit ownership of the restaurant, only if no biological heir appeared two years prior to the latter's death. After his lawyer verifies that Linguini is Gusteau's son, Skinner hides the evidence in an envelope. Some of Emile's friends ask Remy for food from the restaurant, but Remy finds the pantry has been locked. He goes into Skinner's office to find the key, and finds both Gusteau's will and Renata's letter. Skinner arrives and chases after Remy, who has grabbed the letter and the will. Remy manages to escape and gives the documents to Linguini, who becomes the new head of the restaurant and fires Skinner. Linguini and Colette even begin to develop a romantic bond, leaving Remy feeling left out and taken for granted.
Anton Ego learns Gusteau's has become popular again, and shows up to tell Linguini that he will be dining in to review the restaurant. After Linguini takes credit for Remy's cooking at a press conference, he and Remy have a falling out. As revenge, Remy leads the entire clan to raid the restaurant's pantries for food. Linguini arrives to apologize, but upon discovering the raid, he furiously kicks Remy and the other rats out.
The next day, Remy is captured by Skinner. In the cage, Remy has one final conversation with his phantom Gusteau, who tells him that he never needed his guidance and at that moment, he is freed by Django and Emile. After returning to the restaurant, he and Linguini reconcile, and Linguini reveals the truth to his staff, who all immediately quit.
Django organizes the rest of the pack to help out in the kitchen. They throw Skinner and a health inspector, bound and gagged, into the freezer when they try to interfere. Colette returns to the restaurant after seeing Gusteau's "Anyone Can Cook" book. Linguini uses roller skates to wait on all the tables by himself, while Remy and Colette work together to prepare a variation on ratatouille for Ego. Ego is amazed by the dish, which evokes childhood memories of his mother's cooking, and asks to see the chef. Linguini and Colette wait until all the other customers leave to introduce Remy to Ego. Ego writes a glowing review of the meal the next day about how he truly understands Gusteau's motto, and declares Remy (without revealing his name and that he is a rat) as "nothing less than the finest chef in France."
After Skinner and the health inspector are released, they inform the authorities about the rats found in the kitchen. The restaurant is shut down and Ego loses his job and credibility. However, he eagerly invests in a new bistro run by Linguini and Colette, called La Ratatouille, with a kitchen designed for Remy to continue cooking, and a hidden dining area for the rat colony in the attic.
