Vincent Kartheiser was born in Minneapolis, Minnesota, the son of Janet Marie and James Ralph Kartheiser. He made his screen debut with a role in the 1993 film Untamed Heart opposite Marisa Tomeiand Christian Slater. Kartheiser found himself cast in mostly family fare, most notably the action-adventure drama Alaska, in which he starred opposite Thora Birch and Charlton Heston (whose son directed the film). The film led to Kartheiser getting the lead role in Masterminds, playing opposite Patrick Stewart.

Kartheiser next portrayed Bobby, a drug-addled, homeless street urchin in Larry Clark's Another Day in Paradise. Among his cast-mates were James Woods and Melanie Griffith The film earned the young actor significant accolades, and he went on to appear in Strike! (later re-titled All I Wanna Do), opposite Kirsten Dunst Gaby Hoffmann, and Monica Keena the last of whom co-starred with him again in his next project, Crime and Punishment in Suburbia.

Screened at the 2000 Sundance Film Festival, Crime and Punishment in Suburbia was a moody, updated take on Dostoyevsky's Crime and Punishment, set in a modern high school. Kartheiser later landed a regular role on the supernatural drama series Angel in 2002. He played Connor, the son of two vampires, Darla (Julie Benz and the titular Angel (David Boreanaz). Upon returning to L.A., Connor's gradually unraveling mental state was among the main focuses of the show's fourth season (he would make two more appearances during its final season).

Returning to film work, Kartheiser starred in the well-received coming-of-age drama Dandelion, which was shown at the 2004 Sundance Film Festival. He has a supporting role in the television series Mad Men, which debuted on AMC in 2007.

Together with the rest of the cast of "Mad Men," he was nominated for the Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Drama Series in 2008. He lists Monica Keenaas his favorite actress, and has worked with her on two films, Crime and Punishment in Suburbia and Strike!.