The start of Bernie Mac's career was a stand-up comedian in Chicago. Bernie Mac won the Miller Lite Comedy Search in 1990 and his popularity as a comedian began to grow. He opened for Dionne Warwick, Redd Foxx and Natalie Cole and had a number of HBO comedy specials, which helped increase his popularity further.

Later Mac also began acting in minor roles, and received his big break as Pastor Clever in Ice Cube's 1995 film, Friday. Following that role, Bernie Mac would also work in many other movies, and some televison appearances, including Booty Call, How to Be a Player, Life and What's the Worst That Could Happen?. Bernie Mac also had a small recurring role on the sitcom Moesha, playing Moesha's uncle Bernie and the charcater "Andell's" one time Love interest. Bernie Mac was also one of the few African American comedic actors to be able to break out of the traditional "black comedy" genre, having roles in the remake of Ocean's Eleven and becoming the new Bosley for the Charlie's Angels sequel, Charlie's Angels: Full Throttle.

In 1997, Mac continued with his stand-up comedy roots, touring the country as one of the Kings of Comedy, along with Steve Harvey, Cedric the Entertainer and D.L. Hughley. The comedy act would later be filmed by Spike Lee for the movie The Original Kings of Comedy.

In 2001, Fox gave Bernie Mac his own sitcom called The Bernie Mac Show. The show, based on his own life, in which he suddenly becomes custodian over his sister's three children, has been a huge success in part because it allows Mac to stay true to his stand-up comedy roots, breaking the fourth wall to communicate his thoughts to the audience. Mac's show also stars three young actors who play his sister's children. They are Vanessa, Jordan, and Bryana. Bernie plays a fictional version of himself on the show, along with a beautiful young wife named Wanda (Kellita Smith). Much of the comedy arises from Bernie's habitual frustration with his nephew, Jordan (Jeremy Suarez), and his niece, Vanessa (Camille Winbush). This show contains many parodies to events in Bernie's actual life.

In 2004, Mac had his first starring role as a retired baseball player in the movie Mr. 3000.

In February 2005, Mac announced that he has sarcoidosis, a rare autoimmune disease that causes inflammation of the body's tissues, frequently in the lungs. In the announcement, he said that he has suffered from the disease since 1983.

He is number 72 on Comedy Central's list of the 100 greatest standups of all time.