Barton MacLane graduated from Wesleyan University, where he displayed a notable aptitude for sports, in particular football and basketball. Not surprisingly, his physical prowess led to an early role in The Quarterback (1926) with Richard Dix. MacLane once commented that, as an actor, he needed to have the physical strength to tear the bad guys "from limb to limb", if necessary. Ironically, it was usually Barton himself who was destined to be at the end of a hiding (when not getting shot, instead), typically as snarling henchmen, outlaws and other assorted dubious or abrasive types throughout most of his 40-year acting career. In fact, Barton became so typecast that his name was for a time used proverbially, to generally describe a shouting, hard-nosed ruffian.

After training at the American Academy of Dramatic Arts, MacLane joined a stock company in Brooklyn. In 1927 he had his first part on Broadway, a brief moment as an assistant district attorney, in the melodrama "The Trial of Mary Dugan". He then played a small featured role as a police officer in "Subway Express" (1929-30), a drama enacted in the interior of a subway car. In mid-1932 MacLane tried his hand at writing his own starring vehicle for the stage, entitled "Rendezvous". While the play closed after just 21 performances, it led to a contract with Warner Brothers.