Horton was born in Brooklyn, New York, to Isabella S. Diack and Edward Everett Horton. His mother was born in Matanzas, Cuba to Mary Orr and George Diack, immigrants from Scotland. Horton attended the Baltimore City College high school in Baltimore, Maryland, where he was inducted into that school's Hall of Fame. He then attended college at Brooklyn Polytechnic and Columbia University, where he was a member of Phi Kappa Psi Fraternity.
Horton started his stage career in 1906, singing and dancing and playing small parts in Vaudeville and in Broadway productions. In 1919, he moved to Los Angeles, California, and started getting roles in Hollywood films. His first starring role was in the 1922 comedy film Too Much Business, and he portrayed the lead role of an idealistic young classical composer in Beggar on Horseback in 1925. In the late 1920s he starred in two-reel silent comedies for Educational Pictures, and made the transition to talking pictures with Educational in 1929. As a stage-trained performer, he found more movie work easily, and appeared in some of Warner Brothers' early talkies, including The Hottentot and Sonny Boy.
Horton originally went under his given name, Edward Horton. His father persuaded him to adopt his full name professionally, reasoning that there might be other actors named Edward Horton, but only one named Edward Everett Horton.
Horton's screen character was instantly defined from his earliest talkies: pleasant and dignified, but politely hesitant when faced with a potentially embarrassing situation. Horton soon cultivated his own special variation of the time-honored double take (an actor's reaction to something, followed by a delayed, more extreme reaction). In Horton's version, he would smile ingratiatingly and nod in agreement with what just happened; then, when realization set in, his facial features collapsed entirely into a sober, troubled mask.
Horton starred in many unpretentious comedy features in the 1930s, usually playing a mousy fellow who put up with domestic or professional problems up to a certain point, and then finally asserted himself for a happy ending. The actor is best known, however, for his work as a character actor in supporting roles. Some of his noteworthy films include The Front Page, Trouble in Paradise, Top Hat (one of several Astaire – Rogers movies Horton was in), Holiday, Lost Horizon, Here Comes Mr. Jordan, Arsenic and Old Lace, and Pocketful of Miracles.
Horton continued to appear in stage productions, often in summer stock. His performance in the play "Springtime for Henry" became a perennial in summer theaters.
In 1945-47 Horton hosted radio's Kraft Music Hall. During the 1950s, Horton worked in television. One of his most famous appearances is an I Love Lucy episode, where he is cast against type as a frisky, amorous suitor. (Horton, a last-minute replacement for another actor, received a special, appreciative credit in this episode.) Beginning in 1959 he narrated the "Fractured Fairy Tales" segment of the Rocky & Bullwinkle cartoon show. In 1965 he played the medicine man, Roaring Chicken, in the sitcom F Troop. He parodied this role, portraying "Chief Screaming Chicken" on Batman as a pawn to Vincent Price's Egghead in the villain's attempt to take control of Gotham City. His last role, as a moribund tobacco company president in a wheelchair, was in the motion picture Cold Turkey, released after his death.
Edward Everett Horton died of cancer at age 84 in Encino, California. Shortly after he died, the city of Los Angeles, California renamed a portion of Amestoy Avenue, the dead-end street where he lived in the district of Encino, "Edward Everett Horton Lane"