Berkeley was born in Brooklyn, New York on December 16, 1955, but has lived most of his life in New Jersey. He attended Hampshire College and worked in the theaters at the five college system which Hampshire was a part of, including Smith, Mount Holyoke, Amherst, and the University of Massachusetts. Berkeley worked in the Regional and Repertory Theaters in addition to Off Broadway while living in New York. A casting agent saw Berkeley in a play written by Reynolds Price called Early Dark, and encouraged him to move to Hollywood.
Berkeley began playing roles in 1981, with early appearances in M*A*S*H, Cagney & Lacey, Remington Steele, Miami Vice, Moonlighting and The A-Team. Although not becoming a household name, Berkeley's face was increasingly recognizable into the 1990s. His later television guest roles included The X-Files, CSI, ER and Law & Order.
On the big screen, Berkeley has appeared in North Country, Terminator 2: Judgment Day, Mommie Dearest, Phoenix, Kick-Ass, A Few Good Men, The Rookie, Candyman, Apollo 13, Leaving Las Vegas, Gattaca, The Rock (uncredited), Air Force One, Sid and Nancy, Spawn, Amistad, Shanghai Noon, Barb Wire and Timecode. Berkeley has the distinction of appearing in both the made-for-television film L.A. Takedown in 1989 and its 1995 critically acclaimed theatrical remake Heat, both directed by Michael Mann. Several of his earlier roles were in films by director Alex Cox.
In 2001, Berkeley became a recurring guest star and later on as a series regular on 24 in the role of George Mason, the head of the Counter Terrorist Unit. He portrayed the mysterious John Smith on the CBS drama Jericho.
In 2010, he received one of his best known roles - that of Percy Rose in the CW action-thriller series Nikita. He portrayed the character as a series regular in the show's first two seasons as well as being the series' de facto main antagonist of its first two seasons. Berkeley was Sheriff Thomas "Red John" McAllister on The Mentalist.
Berkeley portrays Gregory on AMC's The Walking Dead, originally in a guest role in season 6, but was promoted as a series regular for season 7.
Berkeley has provided voices for animated series such as Aaahh!!! Real Monsters, Teen Titans and Gargoyles. He voiced Quentin Beck/Mysterio in The Spectacular Spider-Man, Captain Atom in the animated movie Superman/Batman: Public Enemies and Dr. Kirk Langstrom in Son of Batman.
In 2013, Berkeley won the Streamy Award "Best Male Performance, Drama" for his starring role in the acclaimed webseries, The Booth at the End.
Berkeley is a painter and a sculptor, as well as a make-up artist. Berkeley met Sarah Clarke on the set of 24 in 2001, and married her the following year. Berkeley and Clarke live with their daughters, Olwyn Harper (born in 2006) and Rowan Amara (born in 2010), in Maine and in Los Angeles, California, where he works as a painter and sculptor when he is not acting.