In Paris in 1910, mother cat Duchess and her three kittens, Marie, Berlioz, and Toulouse, live with retired opera diva Madame Adelaide Bonfamille, and her English butler, Edgar. One day while preparing her will with lawyer Georges Hautecourt, Madame declares her fortune to be left to her cats until their deaths, and thereafter to Edgar. Edgar hears this through a speaking tube, and plots to eliminate the cats. Therefore, he sedates the cats by sleeping pills in their food, and enters the countryside to abandon them. There, he is ambushed by two hounds, named Napoleon and Lafayette, and the cats are stranded in the countryside, while Madame Adelaide, Roquefort the mouse, and Frou-Frou the horse discover their absence. In the morning, Duchess meets an alley cat named Thomas O’Malley, who offers to guide her and the kittens to Paris. The group briefly hitchhike in a milk cart before being chased off by the driver. Later, while crossing a railroad trestle, the cats narrowly avoid an oncoming train, but Marie falls into a river and is saved by O’Malley; himself rescued by two English geese, Amelia and Abigail Gabble, who accompany the cats to Paris. Edgar returns to the country to retrieve his possessions from Napoleon and Lafayette, as the only evidence that could incriminate him.
Travelling across the rooftops of the city, the cats meet O’Malley’s friend Scat Cat and his musicians, who perform the song Ev’rybody Wants to Be a Cat. After the band has departed, O’Malley and Duchess converse on a nearby rooftop while the kittens listen at a windowsill. Here, Duchess’s loyalty to Madame prompts her to decline O’Malley’s proposal of marriage. Duchess and the kittens return to Madame’s mansion, but Edgar places them in a sack and prepares to ship them to Timbuktu; whereupon they direct Roquefort to retrieve O’Malley. He does so, and O’Malley returns to the mansion, ordering Roquefort to find Scat Cat and his gang. This done, the alley cats and Frou-Frou fight Edgar, while Roquefort frees Duchess and the kittens. At the end of the fight, Edgar is locked in his own packing-case and sent to Timbuktu himself. Madame Adelaide’s will is rewritten to exclude Edgar, with Madame expressing surprise at Edgar’s departure. After adopting O’Malley into the family, Madame establishes a charity foundation housing Paris’s stray cats (represented by Scat Cat and his band, who reprise their song).