A husband, Jim Dear, gives his wife Darling an American cocker spaniel puppy which she names Lady. As she grows up, Lady befriends two other dogs; Jock, a Scottish terrier, and Trusty, a bloodhound. Lady also becomes piqued by the presence of a rat, which she keeps from entering the house. After Lady's owners becoming unexpectedly harsh towards her, Jock and Trusty inform her that Darling is most likely expecting a baby. However, a stray dog named Tramp enters the backyard and tells Lady that her owners will soon favor the baby over her, to which Jock and Trusty order him to leave.
Darling gives birth to the baby and the infant is presented to Lady. Soon afterwards, the couple leave for a trip, leaving their Aunt Sarah to look after Lady. Sarah's pet Siamese cats, Si and Am, cause mischief throughout the house, but Sarah blames Lady for the ruckus and takes Lady to a pet shop to buy a muzzle, which she places on Lady's nose. The terrified Lady flees and encounters a pack of stray dogs, which chase Lady and corner her in an alley. However, Tramp arrives and fights the pack, chasing them off. Tramp later on has a zoo beaver remove the muzzle. Lady and Tramp become smitten with each other at an Italian restaurant, but Lady chooses to return home. Tramp accompanies her, but chases several chickens at a barn, resulting in Lady being thrown in the pound. Aunt Sarah reclaims Lady and ties her to a tree at the backyard. Tramp attempts to apologize to Lady, but Lady angrily confronts him and orders him to leave.
Tramp sadly complies and leaves the backyard, but shortly afterwards, the rat returns and sneaks into the house. Lady calls Tramp, who enters the house and fights the rat in the nursery, ultimately killing it. However, Aunt Sarah assumes Lady and Tramp were responsible and locks Lady in the basement before calling the dog-catcher to take Tramp to the pound. Jim and Darling return home and release Lady, who shows them the dead rat. Jock and Trusty overhear the commotion and realize Tramp was only trying to protect the baby and chase after the dog-catchers wagon, ultimately crashing it and allowing Lady to reach Tramp. However, Trusty is injured in the crash. Tramp is later adopted by the family, and Lady gives birth to four puppies, three resembling her and one resembling Tramp. Jock and Trusty attend the house, Trusty with his leg in a cast. Aunt Sarah also sends the dogs cookies, hinting at a reformation.
- Barbara Luddy as Lady.
- Larry Roberts as Tramp.
- Bill Thompson as Jock/Bull/Dachsie/Policeman/Joe.
- Bill Baucom as Trusty.
- Stan Freberg as Beaver.
- Verna Felton as Aunt Sarah.
- Alan Reed as Boris.
- Peggy Lee as Darling/Si and Am/Peg.
- Thurl Ravenscroft as Al.
- Mel Blanc as The Rat.
- George Givot as Tony.
- Dallas McKennon as Toughy/Pedro/Professor/Hyena.
- The Mellomen as Dog Chorus.