In 1935, McIntire married Actress Jeanette Nolan, and the couple had two children together, one of whom was the actor Tim McIntire (1944–1986) who starred in the 1978 film American Hot Wax. Their daughter Holly was also an Actress, appearing in two episodes of Wagon Train. McIntire and Nolan both appeared in Psycho. He played a sheriff, while she voiced some of the "mother" lines. In the Wagon Train episode "The Janet Hale Story" McIntire and Nolan played husband and wife Chris and Janet Hale. In The Virginian, they also played husband and wife. They both appeared again as husband and wife in The Fugitive (1966) season 3, episode 24, as farm workers with their real life son, Tim. In the 1984 comic spy adventure Cloak & Dagger, they again played a couple. This time they portrayed sinister spies posing as harmless elderly tourists.

John McIntire was born in Spokane, Washington and was of Irish descent. He grew up around ranchers, an experience that would later inspire his performances in dozens of film and television westerns. A graduate of the University of Southern California, McIntire began acting in radio on Tarzan and the Diamond of Asher, where he met his Future wife, Jeanette Nolan. McIntire worked on many episodes of Suspense from the early 1940s. He was the narrator for the radio program The March of Time. He was active in the theatre, before he embarked on a lengthy film and television career as a character actor. He was already 40 when he made his big-screen debut in 1947, but went on to appear in films, often portraying police chiefs, judges, eccentric loners or other western characters.

In the mid-1950s, McIntire moved into television, appearing in anthology series, sitcoms and dramas. He guest-starred as Judson in the episode "Chinese Invasion" of NBC's one-season western series, Cimarron City, with George Montgomery and John Smith.

Though he technically played a supporting part, McIntire received top billing and his greatest critical acclaim for the fact-based crime movie The Phenix City Story (1955). He played a real-life reform Politician who was assassinated by the Mob.

In 1960, McIntire guest-starred as william Palmer in the series finale, "The Most Dangerous Gentleman", of the short-lived NBC western Overland Trail, starring william Bendix and Doug McClure, his subsequent co-star on The Virginian. In 1967, he guest-starred in an episode of CBS's short-lived western, Dundee and the Culhane.

Though McIntire had never played the lead in a theatrical film, television provided him with his most prominent and long-running role when in 1961 he replaced the late Ward Bond in the popular NBC/ABC series Wagon Train, playing trailmaster Chris Hale in more than 150 episodes between 1961 and 1965. His co-stars were Robert Horton, Robert Fuller, Denny Scott Miller, Terry Wilson, Frank McGrath, and Michael Burns.

McIntire replaced actor Charles Bickford (who had himself replaced Lee J. Cobb) on NBC's The Virginian in 1967 when Bickford abruptly died. McIntire played the role of the brother of Bickford's character for four seasons, a major recurring leading role in a weekly 90-minute western series similar in size and scope to his earlier work on Wagon Train.

McIntire and Nolan also worked together as voice actors. In a 1969 KCET television reading of Norman Corwin's 1938 radio play The Plot to Overthrow Christmas, McIntire played the Devil and Nolan played Lucrezia Borgia. In 1977 they appeared in the Disney animated film The Rescuers, in which he voiced the cat Rufus and she the muskrat Ellie Mae. Four years later, the couple worked on another Disney film, The Fox and the Hound, with McIntire as the voice of Mr. Digger, a badger, and Nolan as the voice of Widow Tweed.

He played the supporting role of Judge Parker in Rooster Cogburn (1975), the sequel to True Grit starring John Wayne and Katharine Hepburn, and appeared as Owen Keating in the 1977 television miniseries Aspen. His final film role was in Turner & Hooch (1989).

John McIntire died on January 30, 1991 from emphysema and lung cancer in Pasadena in Los Angeles County. Aside from Nolan, he was also survived by their daughter, Holly McIntire-Wright and a grandson Luke Wright. McIntire and Nolan's son, actor/musician Tim McIntire, predeceased his parents, having died in 1986 from heart problems.