Kirshner found a talent agent at the age of 12, and was acting professionally by the age of 15. She made her film debut in 1993 in Denys Arcand’s Love and Human Remains. She convinced her father to sign a “nudity waiver” to play a dominatrix. Kirshner won a Genie nomination for Best Performance by an Actress in a Supporting Role.
The following year, she starred in Atom Egoyan’s Exotica. In 1996, she appeared in The Crow: City of Angels. Kirshner also appeared in the first three episodes of 24 as the bisexual assassin Mandy in 2001. She would later reprise the role for the second season’s finale and in the latter half of the show’s fourth season. Also in 2001, Kirshner played Catherine Wyler, The Cruelest Girl in School, in Not Another Teen Movie. The character is primarily a spoof of Kathryn Merteuil (played by Sarah Michelle Gellar) in Cruel Intentions, and was partially based on Mackenzie Siler (played by Anna Paquin) from She’s All That.
In Marilyn Manson’s music video for “Tainted Love”, which was featured on the movie’s soundtrack, she made a cameo appearance as her character Catherine Wyler. In 2004, Kirshner was cast as author Jenny Schecter, a main character in the drama series The L Word. She remained with the show for all of the show’s six seasons through 2009.
In 2006, she starred in Brian De Palma’s The Black Dahlia in which she plays the young aspiring actress, Elizabeth Short, who was mysteriously mutilated and murdered in 1947. While the film itself was critically panned, many reviews singled out her performance for acclaim. In 2010, Kirshner co-starred in the film 30 Days of Night: Dark Days which began filming in the fall of 2009. In 2010, she was cast as Isobel Fleming, a guest role on The Vampire Diaries.