Laden with laughs, this upbeat animation adventure revolves around Remy, an idealistic and ambitious young rat, gifted with highly developed senses of taste and smell. Inspired by his idol, the recently deceased chef Auguste Gusteau, Remy dreams of becoming a cook himself. When an old woman sees his clan, they are forced to abandon their home; Remy is separated from them as a result of the woman's gunshots. He ends up in the sewers of Paris and eventually finds himself at a skylight overlooking the kitchen of Gusteau's restaurant.
As Remy watches, a young man named Alfredo Linguini, son of Gusteau's former Italian girlfriend Renata Linguini, is hired as a garbage boy by Skinner, the restaurant's devious current owner and Gusteau's former sous-chef. When Linguini spills a pot of soup and attempts to recreate it with disastrous results, Remy falls into the kitchen and uses other ingredients to complement the soup to perfection.
Linguini catches Remy and is confronted by Skinner. As Skinner yells at Linguini, the soup is accidentally served and proves to be a success. Colette Tatou, the staff's only female chef, convinces Skinner to retain Linguini, who is assumed to be the soup's creator. After Skinner catches Remy in the act of escaping, he orders Linguini to take the rat far away and kill it. Linguini then discovers Remy's intelligence and passion for food, so he keeps him.
Remy discovers that he can control Linguini's movements by pulling his hair.
On Linguini's first day as a chef, he and Remy find a way to communicate; Remy guides Linguini like a marionette by pulling on his hair while hidden under Linguini's toque blanche, while Skinner assigns Colette to train his new cook.
Suspicious, Skinner learns that the boy is Gusteau's illegitimate son and the rightful heir to the restaurant, which threatens his use of the restaurant's reputation to establish a packaged food franchise he started after Gusteau died. Remy discovers the evidence of Linguini's inheritance and, after eluding Skinner, gives it to Linguini, who deposes Skinner as owner.
The restaurant continues to thrive, and Linguini and Colette develop a budding romance, leaving Remy feeling left out. Meanwhile, Remy reunites with his father, Django, and his brother, Emile, who take him back to their new lair. Though thrilled that his family and clan are safe, he tells him that he cannot stay. In an attempt to rid his son from his like of humans, Django shows Remy a window display of dead rats, poison, and traps. Remy refuses to listen and leaves.
France's top restaurant critic Anton Ego, whose previous review cost Gusteau's one of its star ratings (and the heartbroken chef's life) announces he will be re-reviewing the restaurant the following evening. After a falling-out with Linguini, Remy leads his clan in a raid on the restaurant's pantries. Linguini catches them and throws them out. Skinner, now aware of Remy's skills, captures him in an attempt to use him to create a new line of frozen foods.
However, Remy is freed by Django and Emile. He returns to the restaurant, only to find Linguini is unable to cook without him. Linguini apologizes and reveals the truth to the staff, who all walk out in disgust. Colette later returns after recalling Gusteau's motto, 'Anyone can cook.'
Django arrives with the rest of the clan, offering to help after seeing his son's determination. Remy directs the rats, while Linguini serves as waiter. For Ego (and Skinner), Remy and Colette create a variation of ratatouille, which brings back an astonished Ego memories of his mother's cooking. During the service, the rats are forced to kidnap and tie up Skinner and a health inspector to prevent them from revealing their involvement in the cooking.
When Ego requests to see the chef, Linguini and Colette make him wait until the rest of the diners have left before introducing Remy. Ego is stunned and leaves the restaurant, deep in thought. He writes a positive and thoughtful review for the newspaper the next day, stating that Gusteau's chef (Remy) is 'nothing less than the finest chef in France.'
Despite the positive review, Gusteau's is closed down, since Linguini and Remy had no choice but to release Skinner and the health inspector. Ego loses all credibility as a critic but funds a popular new bistro, "La Ratatouille", created and run by Remy, Linguini, and Colette; Ego frequents the bistro for Remy's cooking. The rats settle in their new home in the bistro's roof.