The Posts resided at 17230 Valley Spring Road in the San Fernando Valley of Los Angeles.

The Mister Ed show concept was derived from a series of short stories by children's author Walter R. Brooks, which began with The Talking Horse in the September 18, 1937, issue of Liberty magazine. Brooks is otherwise best known for the Freddy the Pig series of children's novels, which likewise featured talking animals that interact with humans. Sonia Chernus, secretary to Director Arthur Lubin, introduced Lubin to the Brooks stories and is credited with developing the concept for television.

The first horse that played Mister Ed for the first, unaired pilot episode was a chestnut gelding. The horse proved to be unruly and difficult to work with and was replaced with the horse named Bamboo Harvester (1949–1970), a crossbred gelding of American Saddlebred, Arabian and grade ancestry. A second pilot episode was filmed and Bamboo Harvester remained with the series until its cancellation.

The show's concept resembles that of the Francis the Talking Mule movies in which an equine title character talks, but only to one person, thus causing a variety of opportunities and frustrations. The first six Francis films (1950–55) were also directed by Lubin.

Comedian George Burns financed the original pilot for Mister Ed which was shot at his McCadden Studio in Hollywood at a cost of $70,000. Scott McKay played Wilbur. Jack Benny was also involved behind the scenes. Unable to sell the show to a network, Lubin decided to sell the show into syndication first. He managed to get single sponsor identification for the program on over 100 stations. The show was recast with Alan Young in the lead. Production began in November 1960, although Lubin did not direct early episodes because he was working in Europe on a film. The first 26 episodes were well received enough for the show to be picked up by CBS.

The series was sponsored from 1961 to 1963 by Studebaker-Packard Corporation and Studebaker Corporation. At first, sponsorship came from Studebaker's dealer association, with corporate sponsorship coming from South Bend once the series had been picked up by CBS. Studebakers were featured prominently in the show during this period. The Posts are shown owning a 1962 Lark convertible, and the company used publicity shots featuring the Posts and Mister Ed with their product (various cast members also appeared in "integrated commercials" for Lark at the end of the program). When another Lark convertible served as the official pace car at the 1962 Indianapolis 500, Connie Hines attended the race as part of the promotion.

Studebaker's sales dropped dramatically in 1961 and, despite their exposure via sponsoring this program, never recovered. Studebaker ended U.S. motor vehicle production on December 20, 1963. Later, Studebaker's sponsorship and vehicle-supply agreement ended, and The Ford Motor Company provided the vehicles seen on-camera starting at the beginning of 1965. (Studebaker vehicle production ended in March 1966.)

Another story claims that by 1968 Bamboo Harvester suffering old age ailments and was euthanized in 1970, with no publicity, and buried at Snodgrass Farm in Tahlequah, Oklahoma.

A different horse who died in Oklahoma in February 1979 was widely thought to be Bamboo Harvester, but this horse was, in fact, a horse that posed for the still pictures of Mister Ed used by the production company for the show's press kits. This horse was unofficially known as "Mister Ed", which led to his being reported as such (including sardonic comments on Saturday Night Live's Weekend Update) following his own death.

The theme song received renewed publicity twenty years after the show went off the air when Jim Brown, a preacher from South Point, Ohio, claimed in May 1986 that it contained "satanic messages" if heard in reverse. Brown and his colleague Greg Hudson claimed that the phrases "Someone sung this song for Satan" and "the source is Satan" would be audible. At their behest teenagers burned over 300 records and cassettes of secular music with alleged satanic messages. The teens did not burn a copy of Television's Greatest Hits, but Brown asserted that "Satan can be an influence whether they [the songwriters] know it or not. We don't think they did it on purpose and we're not getting down on Mister Ed."

A race horse named after the character in the television show took part in the 1994 Grand National steeplechase at Aintree, England but did not complete the course.

In 2004, a remake was planned for the Fox network, with Sherman Hemsley as the voice of Mister Ed, David Alan Basche as Wilbur, and Sherilyn Fenn as Carol.

MGM also released a single-disc release entitled Mister Ed's Barnyard Favorites on July 26, 2005 which contains the first eight episodes featured on Volume One.

In 2007, it was reported that a housing developer intended to create a community near Tahlequah, Oklahoma, built around the supposed final resting place of Mister Ed (who died in 1970). It was intended to be themed to the style of the show and its period.

In 2012, Waterman Entertainment announced they were developing a new feature film based on Mister Ed.

On December 9, 2014, Shout! Factory released Mister Ed- Complete Series on DVD in Region 1. The 22-disc set contains all 143 episodes of the series as well as bonus features.

Shout! Factory announced in June 2009 that they had acquired the rights to release Mister Ed on DVD, and subsequently released the six seasons on DVD in Region 1 in the U.S. Notably, Seasons 4 and 5 are not available outside of the continental U.S. The sixth and final season was released on May 12, 2015.