Born in Toronto, Ontario, she is a granddaughter of Holocaust survivors. Her father, Sheldon, a journalist for The Canadian Jewish News, was born in a displaced person camp in Germany in 1946. He met her mother, Etti, a Bulgarian Jewish refugee, in Israel. Kirshner has a younger sister.

In 1993 she played a dominatrix in Denys Arcand's movie Love and Human Remains. She won a Genie nomination for Best Performance by an Actress in a Supporting Role. The year after she starred in Atom Egoyan's Exotica, and her performance won her a Cannes Critics Choice Award.

Kirshner appeared in the first three episodes of 24 as bisexual terrorist Mandy in 2001. She would later reprise the role for the second seasons finale and in the latter half of the show's fourth series.

Mia Kirshner studied Russian literature and 20th century movie industry at McGill University in Montreal.

In 2006, she starred in Brian De Palma's movie The Black Dahlia, alongside Josh Hartnett, Aaron Eckhart, Scarlett Johansson and Hilary Swank. Kirshner plays a young actress, Elizabeth Short, who was horrifically murdered in 1947.

She has finished working on the book I Live Here. In the book, woman and children refugees from places such as Chechnya, Juárez (Mexico) and Africa will tell their life stories. It will be published in Canada in the spring of 2007. It was supported and funded by Amnesty International to which proceeds from the book will go to.

Kirshner has portrayed Jenny Schecter on the hit Showtime series, The L Word since the show aired in 2004. She is currently reprising her role in season four, which began airing on Showtime, on January 7.

Because she has played many bisexual and lesbian roles, her sexual orientation has been quite a subject of interest. However, when asked in interviews, she always replies ambiguously.