Chambers was born in Springfield, Ohio to Pam and John Chambers. He has a twin brother named Jason, one older brother and two older sisters. He has five children with his wife, Keisha, a former model agency booker which he married in 1993. He and his twin brother visited hospitals frequently as children due to constant bouts with pneumonia. His 5 children are called Isabella, twins Maya and Kaila, Eva and Jackson. His wife and children have recently moved to Los Angeles with Justin from their home in a suburb just outside of New York City. When Justin went on Jimmy Kimmel Live, he mentioned that his daughters have recently formed a band called Nyla!

Chambers was discovered by a modeling scout on the Paris Metro and went on to represent Calvin Klein in a fragrance campaign. He worked throughout Europe, Japan, and the United States for CK, Armani, and Dolce & Gabbana.

His first major acting role came in the music video "Ants Marching" by Dave Matthews Band in 1994. After deciding on an acting career, he moved to New York, where he studied at H.B. Studios for four years. That landed him a few television roles, including Another World and New York Undercover. More recently, he starred in several movies, notably The Wedding Planner (2001, alongside Jennifer Lopez), The Musketeer (2001) Southern Belles (2005), The Zodiac (2005), and the HBO original movie Hysterical Blindness (2002).

Chambers was cast as one of the leads in the CBS crime drama Cold Case but left after appearing in only three episodes. Now he plays intern Alex Karev on the hit ABC medical show Grey's Anatomy.