In Paris 1910, mother cat Duchess and her three kittens, Berlioz, Marie, 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 that her fortune will 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. He later sedates them by putting sleeping pills in a milk mixture intended for them, and drives them to the countryside to abandon them. There, he is ambushed by two hounds named Napoleon and Lafayette, losing his hat and umbrella, 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 hitchhikes in a milk truck before being chased out 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, who in turn has to be rescued by two English geese, Amelia and Abigail Gabble, who accompany the cats to Paris. Meanwhile, Edgar returns to the country to retrieve his possessions from Napoleon and Lafayette, after realising that they are 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, and Duchess’ loyalty to Madame prompts her to decline O’Malley’s marriage proposal. The next day, Duchess and the kittens return to Madame’s mansion, but Edgar finds them before she does, places them in a sack and prepares to ship them to Timbuktu. Roquefort catches up with O’Malley at the cats’ instruction, and O’Malley returns to the mansion, sending Roquefort to find Scat Cat and his gang; while he struggles to explain why he was sent to find them, Roquefort successfully brings them to the mansion. 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, never to be seen again. The cats return to Madame Adelaide, whose will is rewritten to exclude Edgar, with Madame remaining ignorant of the reason for his departure. After adopting O’Malley into the family, Madame establishes a charity foundation housing Paris’ stray cats (represented by Scat Cat and his band, who reprise their song).