MacLane was born in Columbia, South Carolina, on Christmas Day, 1902. He attended Wesleyan University in Middletown, Connecticut, where he excelled at American football. His first movie role, in The Quarterback (1926), was a result of his athletic ability. He then attended the American Academy of Dramatic Arts.

He made his Broadway debut in 1927, playing the assistant district attorney in Bayard Veiller’s The Trial of Mary Dugan. He then performed in the 1928 Broadway production of Gods of the Lightning and was part of the original cast of Subway Express as Officer Mulvaney in 1929. He appeared in the Marx Brothers’ 1929 film debut The Cocoanuts. MacLane made his first credited film appearance in the 1931 romantic drama His Woman. The following year, he wrote the play Rendezvous, which he sold to Arthur Hopkins. The play was performed on Broadway, with MacLane in a featured role.

Maclane played several musical instruments, including the violin, piano, and guitar. In 1939, MacLane married actress Charlotte Wynters. From the 1940s until his death, he maintained a cattle ranch in eastern Madera County, California, where he made his home when he was not acting. He adopted a daughter.

In the 1950s, MacLane began to appear regularly on television. Between 1953 and 1967, he guest starred on such programs as Conflict, Lux Video Theatre, Westinghouse Desilu Playhouse, Laramie, The Monkees, and Gunsmoke. In 1958 he played Sen. Harriman Baylor in the Perry Mason episode, “The Case of the Foot-Loose Doll”. In 1960 he played Eugene Norris, Perry’s friend and small-town sheriff, in “The Case of the Violent Village”. In 1964 he played Archer Osmond in “The Case of the Ruinous Road”.

MacLane’s many other film credits during the 1940s include The Big Street, Victor Fleming’s Dr. Jekyll and Mr Hyde, Fritz Lang’s Western Union, Reginald Le Borg’s The Mummy’s Ghost, and Frank Borzage’s The Spanish Main. He also appeared in two Tarzan films starring Johnny Weissmuller, Tarzan and the Amazons and Tarzan and the Huntress. Some of MacLane’s films during the 1950s include Kiss Tomorrow Goodbye, The Glenn Miller Story, and Three Violent People. In 1955 MacLane appeared as Jim Mablett in the movie Foxfire co-starring Jane Russell.

In 1965, MacLane was cast in the recurring role of General Martin Peterson on I Dream of Jeannie. He appeared in 35 episodes of the series between 1965 and 1969. Three of MacLane’s episodes were aired after his death in January 1969. His character was replaced on later episodes of the series by General Winfield Schaeffer, portrayed by Vinton Hayworth, until his death in 1970. Coincidentally, Hayworth also died before all episodes featuring his character were broadcast.