For his role as William Bradford in Saints & Strangers he was nominated for the Critics' Choice Television Award for Best Actor in a Miniseries or Movie.

Some of Kartheiser's first acting experiences were on stage of Children's Theatre Company in Minneapolis, including roles in Pippi Longstocking, Our Town, Dr. Seuss' The 500 Hats of Bartholomew Cubbins, and Rebecca of Sunnybrook Farm.

Kartheiser found himself cast in mostly family fare, including The Indian in the Cupboard (1995) and a starring role in the film Alaska (1996).

Kartheiser played the role of ambitious young ad man Pete Campbell in the AMC television series Mad Men (2007–2015).

Along with the rest of the show's cast, Kartheiser won the Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Drama Series in 2009 and 2010.

[11] In the summer of 2013, he returned to his theatrical roots in Minneapolis to portray Mr. Darcy in an adaptation of Pride and Prejudice at the Guthrie Theater.

Kartheiser was the subject of at least two complaints and two internal investigations by Warner Bros Television over allegations of disruptive, juvenile behavior and inappropriate comments on set.

In a 2010 interview with The Observer, Kartheiser revealed that he eschewed the extravagant lifestyle of a Hollywood celebrity in favor of simple living.