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.

During the 1930s and 1940s, MacLane worked alongside Humphrey Bogart in several films. He played Lieutenant Dundy opposite Bogart's Sam Spade in The Maltese Falcon, writer/director John Huston's acclaimed film noir based upon Dashiell Hammett's novel. MacLane again collaborated with both Bogart and Huston on the Academy Award-winning 1948 adventure film The Treasure of the Sierra Madre.

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.

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 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".

During the 1960-1961 television season, MacLane was a series regular on twenty-seven episodes of NBC's western, Outlaws, in which he played Marshal Frank Caine. He made his last film appearance in Frank Capra's Academy Award-nominated 1961 comedy Pocketful of Miracles.

MacLane was cast in 1965 in the recurring role of General Martin Peterson on I Dream of Jeannie. He appeared in thirty-five episodes of the series between 1965 and 1969. Three of MacLane's episodes were aired after his death. His character was replaced on later episodes of that show by Gen. Winfield Schaeffer, played by Vinton Hayworth, who died the following year. Hayworth also died before all episodes featuring his character had aired.

MacLane died of double pneumonia on New Year's Day, 1969 in Santa Monica, California. He was buried in Valhalla Memorial Park Cemetery. His survivors included his wife, an adopted daughter, and a grandchild.