
Remy is a rat who lives in the attic of a French country home with his brother Emile and a pack led by his father Django. Gifted with a keen sense of smell and taste, Remy aspires to be a gourmet chef, inspired by France's recently deceased top chef, Auguste Gusteau, but instead he is put to work sniffing for rat poison. When the pack is discovered by the home's occupant, they flee into the sewers; Remy is separated and ends up aground underneath Gusteau's restaurant in Paris.
Led by his imagination of Gusteau, Remy goes to the kitchen skylight to watch the kitchen in action during a service. There, he observes Alfredo Linguini, the son of Gusteau's former mistress, being hired as a janitor by Skinner, the restaurant's current owner and Gusteau's former sous-chef. When Linguini spills some of the soup and attempts to recreate it using random ingredients, Remy is horrified and falls into the kitchen; instead of escaping, Remy attempts to correct the soup. Remy is caught by Linguini just as Linguini is caught by Skinner, but before anyone can stop the serving staff, the soup is served and found to be a success. Colette, the staff's only female chef, convinces Skinner to retain Linguini, believing him to be the success behind the soup. Linguini takes Remy home, realizing he cannot kill him as instructed by Skinner as Remy was the "little chef" that made the soup.
Remy discovers that he can control Linguini's movements by pulling on his hair.
Remy and Linguini find a means to overcome their language barrier, with Remy pulling Linguini's hair under his toque blanche to control his limbs like a marionette. The two work together to successfully cook in the kitchen, even overcoming challenges placed by Skinner. Skinner, suspicious of Linguini's talents, discovers that Linguini is actually Gusteau's son and by Gusteau's will, is the rightful owner of the restaurant; this revelation would ruin Skinner's plans to use Gusteau's name to market a line of microwaveable meals. Remy discovers Skinner's documents and retrieves them, bringing them to Linguini, who subsequently fires Skinner and takes control of the restaurant, much to the staff's delight. Linguini and Colette even begin to develop a romantic bond, with Remy feeling that he is being left behind. Remy finds Emile in the restaurant's trash, and is reunited with the pack. Django warns Remy that humans and rats will never get along, but Remy insists that it will all work out. Meanwhile, Remy begrudgingly feeds Emile and his growing group of friends from the kitchen's pantry as the nights pass.
Anton Ego, a food critic that had cost Gusteau's one of its star ratings, announces he will review the restaurant again the next day based on its rising success. Linguini, under pressure of Ego's pending arrival, has a fallout with Remy, causing Remy to retaliate by leading a raid on the kitchen's foodstocks that night. Linguini catches the rats stealing the food and chases them all out, including Remy, telling the rat he never wants to see him again. Remy, dejected, is captured by Skinner who recognizes Remy was the real talent behind Linguini, but is later freed by Django and Emile. Remy returns to the kitchen to find a frantic Linguini apologizing for his actions, and asking Remy back to help. Linguini then reveals the truth to the rest of the staff about Remy, causing them all to walk out, but Colette later returns after recalling Gusteau's motto: "Anyone can cook." Django, impressed by his son's determination, organizes the rest of the pack to help out in the kitchen to serve the other customers along with Linguini, while Remy and Colette work together to prepare a variation on ratatouille for Ego. Ego is amazed by the dish, bringing back memories of his mother's cooking, and asks to see the chef. Linguini and Colette wait until all the other customers leave to reveal Remy and the rats to Ego. Ego leaves the restaurant deep in thought, and writes a glowing review of the meal the next day, declaring Remy to be "nothing less than the finest chef in France".
Gusteau's restaurant is closed a short time later after a health inspector discovered the rats in the kitchen. Though Ego's reputation is tarnished on reviewing a restaurant plagued with rats, he eagerly funds a popular new bistro, "La Ratatouille", run by Linguini and Colette, featuring diner areas for both humans and rats and a kitchen designed for Remy to continue to cook in.