Glover originally worked in city administration. In his late 20s, he enrolled in the Black Actors Workshop at the American Conservatory Theater, a regional training program in San Francisco. Glover also trained with Jean Shelton at the Shelton Actors Lab in San Francisco. In an interview on Inside the Actor's Studio, Glover credited Jean Shelton for much of his development as an actor. Deciding that he wanted to be an actor, Glover resigned from his city administration job and soon began his career as a stage actor. Glover then moved to Los Angeles for more opportunities in acting, where he would later go on to co-found the Robey Theatre Company with actor Ben Guillory in honor of the actor, radical activist, and concert singer Paul Robeson in Los Angeles in 1994.

Glover has had a variety of film, stage, and television roles, and is best known for playing Los Angeles police Sergeant Roger Murtaugh in the Lethal Weapon series of action films. During his career, he has made many cameo appearances. For example, he appeared in the Michael Jackson video Liberian Girl of 1987. He has also appeared as the husband to Whoopi Goldberg's character Celie in The Color Purple, and as Lieutenant James McFee in the film Witness. In 1994 he made his directorial debut with the Showtime channel short film Override. Also in 1994, Glover and actor Ben Guillory formed the Robey Theatre Company in Los Angeles, focusing on theatre by and about Black people.

Glover earned top billing for the first time in Predator 2, the sequel to the sci-fi action film Predator. That same year he starred in Charles Burnett's To Sleep with Anger, for which he won the Independent Spirit Award for Best Male Lead.

In common with Humphrey Bogart, Elliott Gould and Robert Mitchum, who have played Raymond Chandler's private eye detective Philip Marlowe, Glover played the role in the episode "Red Wind" of the Showtime network's 1995 series Fallen Angels.

In addition, Glover has been a voice actor in many children's movies. Glover was featured in the popular 2001 film Royal Tenenbaums, also starring Gwyneth Paltrow, Anjelica Huston, Ben Stiller and Owen Wilson.

In 2004, he appeared in the low-budget horror film Saw as Detective David Tapp. In 2005, Glover and Joslyn Barnes announced plans to make No FEAR, a movie about Dr. Marsha Coleman-Adebayo's experience. Coleman-Adebayo won a 2000 jury trial against the US Environmental Protection Agency (EPA). The jury found the EPA guilty of violating the civil rights of Coleman-Adebayo on the basis of race, sex, color and a hostile work environment, under the Civil Rights Act of 1964. Coleman-Adebayo was terminated shortly after she revealed the environmental and human disaster taking place in the Brits, South Africa, vanadium mines. Her experience inspired passage of the Notification and Federal Employee Anti-discrimination and Retaliation Act of 2002 (No FEAR Act).

In 2009, Glover performed in The People Speak a documentary feature film that uses dramatic and musical performances of the letters, diaries, and speeches of everyday Americans, based on historian Howard Zinn's "A People's History of the United States".

Glover played President Wilson, the President of the United States in 2012, a disaster film directed by Roland Emmerich and released in theaters November 13, 2009.

NOTE: Bio updated on June 14, 2011.