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 & Gabbana1. 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 resident Alex Karev on the hit ABC medical show Grey's Anatomy.

His first major acting role came in the music video "Ants Marching" by Dave Matthews Band in 1994.