The show was based on short stories by Walter R. Brooks that began with 'The Talking Horse' in a 1937 issue of Liberty magazine; Brooks was also known for the Freddy the Pig children's novels featuring talking animals.

Director Arthur Lubin wanted to make a TV series based on his Francis the Talking Mule movies but couldn't secure the rights, so he optioned Brooks' talking horse stories instead; his secretary Sonia Chernus is credited with introducing him to the stories and developing the TV concept.

Comedian George Burns financed the show's pilot for $70,000, shot at his McCadden Studio in Hollywood, with Jack Benny also involved behind the scenes; Lubin failed to sell the show to a network and instead launched it in first-run syndication.

The original unaired pilot, titled 'Wilbur Pope and Mister Ed', starred Scott McKay as Wilbur and Sandra White as his wife, using a script nearly identical to the eventual series premiere before the show was recast with Alan Young and Connie Hines.

Director Arthur Lubin said Alan Young was chosen for the lead because he 'just seemed like the sort of guy a horse would talk to.'

The horse used for the first unaired pilot was a chestnut gelding who proved unruly and difficult to work with, and was replaced by Bamboo Harvester, a crossbred gelding of American Saddlebred, Arabian and grade ancestry, who then played Ed for the entire series run.

To make it look like Ed was talking, trainer Les Hilton initially placed a piece of nylon thread in the horse's mouth, a technique borrowed from the Francis the Mule films; Bamboo Harvester eventually learned to move his lips on cue when the trainer touched his hoof, and even did it whenever Alan Young stopped talking mid-scene.

Alan Young later admitted he invented the popular rumor that peanut butter applied to the horse's gums made it appear to talk, at the suggestion of producers who wanted to keep the real technique secret so children wouldn't be disappointed.

Ed's stablemate, a quarter horse named Pumpkin, served as Bamboo Harvester's stunt double on the show and later appeared again in the TV series Green Acres.

Mister Ed's voice was provided uncredited by former B-movie cowboy star Allan 'Rocky' Lane, who was referred to by producers only as 'an actor who prefers to remain nameless'; after the show became a hit he asked for on-screen credit but accepted a raise instead.

The theme song was written by Jay Livingston and Ray Evans, with Livingston himself singing it after the producers liked his vocals enough to keep them, originally intending to only use his voice until a professional singer was found; Sheldon Allman provided Ed's singing voice for episodes.

In 1986, a preacher from Ohio claimed the Mister Ed theme song contained satanic messages when played backward, leading some teenagers to burn records and cassettes of secular music, though he did not target a copy containing the actual song.

Sharon Tate appeared in a 1963 episode ('Love Thy Neighbor') years before she was murdered by members of the Manson Family in 1969.

Several future stars of other famous shows appeared as guests, including Neil Hamilton (later Commissioner Gordon on Batman), Hayden Rorke (later Dr. Bellows on I Dream of Jeannie), and Alan Hale Jr. (later the Skipper on Gilligan's Island).

The show was sponsored by Studebaker from 1961 to 1963, with the Posts driving a 1962 Lark convertible on screen; despite the promotional tie-in, Studebaker's sales dropped and the company ended U.S. vehicle production in December 1963, after which Ford began supplying the show's cars starting in 1965.

In 1995, Walt Disney Pictures approached Kelsey Grammer to play Wilbur Post in a planned feature film adaptation inspired by Cyrano de Bergerac, but Grammer declined because he didn't want his first major film role to be based on a TV character.