In Paris, France, wealthy and aging socialite Madame Adelaide Bonfamille lives with her cat Duchess, and Duchess' three kittens: Berlioz, Toulouse, and Marie. Also in her employ is a butler, named Edgar.
One day, Madame sends for her friend, George Hautecourt. The task is to finalize and make up Madame's will. Unknown to the two of them, Edgar is listening in. Madame notes that she has no living relatives, and considers her cats to be just as much a part of her family as anything else. As Edgar listens, he's shocked that Madam is planning to give her entire fortune to them, with the estate to be turned over to Edgar once the cats expire.
Of course, Edgar is not at all happy about this, as the cats will easily outlive him! Edgar slips some sleeping pills in the cat's daily serving of cream, and once they have passed out, and night has fallen, he takes them out to the countryside, attempting to abandon them. However, his plans are ruined when a pair of dogs named Lafayette and Napoleon give chase. Edgar abandons the cats in a field before heading back to Paris.
The next day, the cats awaken to find themselves not in their home, but in the countryside, and soon meet a cat named Thomas O'Malley. After some talk, O'Malley offers to help get the family back to Madame Bonfamille.
Meanwhile, back in Paris, a house-mouse in Madam Bonfamille's residence named Rocquefort confirs with Madame's coach horse, Frou-Frou. Both are unsure what has become of their friends, when Edgar comes into Frou-Frou's stables very chipper. As Frou-Frou can't talk, Edgar shows her the headline from the morning's paper, telling of a mysterious catnapper taking off with Duchess and her children. Edgar seems to think he's gotten away with his little crime...until he realizes that he left his hat and umbrella out in the countryside, and quickly rushes out to retrieve them.
Meanwhile, Thomas, Duchess and the kids hitch a ride on a milk truck, before being found out by the driver. They next attempt to follow a train track, until a train causes them to dive off. However, little Marie gets swept up in a nearby river. Thomas jumps in to save her, and the family follows him downstream.
They then meet up with two female geese, Abigail and Amelia. The group then follow the two to their Uncle Waldo. Their journey takes them to a restaurant where Uncle Waldo has escaped from becoming the main course. However, being marinated in white wine has caused Uncle Waldo to be quite tipsy.
Meanwhile, Edgar returns to the countryside and manages to get back his hat and umbrella, but not before Napoleon and Lafayette attempt to nip at his heels again.
Back in Paris, the cats are almost home, but worn out from their trip. O'Malley then takes them to Scat Cat's abode in an abandoned building. Scat Cat and his gang of alley cats entertain the family, before heading off into the night.
After the kittens are put to bed, Duchess and O'Malley share a tender moment. While Duchess tells of her eagerness to return to Madame, O'Malley (who has never really known kindness from humans), just thinks Madame sees Duchess and her kittens as housepets. Duchess however, explains that Madame sees them as more than this: they are the equivalent of a family to her.
The next morning, O'Malley leads the family back to Madame's mansion. After saying his goodbyes, the cats head to the front door...only to be confronted by Edgar, who quickly throws them in a sack.
Rocquefort has seen the whole thing, and after going to Duchess and the kittens in the sack, is told to find O'Malley. Rocquefort runs off and catches up to O'Malley. Thomas heads back to the mansion, and tells Rocquefort to find Scat Cat and his gang.
Back in the mansion's stables, Edgar puts the cats into a trunk, telling them of his plans to send them to Timbuktu. He has already called for a truck to pick the trunk up, when O'Malley bursts in and attacks Edgar. Soon after, Scat Cat and his gang arrive to help, while Rocquefort manages to pick the lock and get the cats out. Scat Cat's gang, with some help from Frou-Frou, manage to knock Edgar into the trunk and out the stable's doors, just as the pick-up service arrives, taking it away.
Some time afterward, Madame Bonfamille finds that Edgar has gone missing for some unknown reason, and calls on George to repurpose her will, removing him from any inheritance. Thomas is welcomed into the family, and Madam also has chosen to allow her mansion to be a safe haven for all the alley cats in Paris.
The film ends with Scat Cat and his gang throwing a wild party at Madame's abode.