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, yet another time showing her talents as an actress. Her performance in Exotica won her a Cannes Critics Choice Award.

After working for a decade in film and television, Kirshner became well known when she starred in the first three episodes of 24 as bisexual terrorist Mandy, in 2001. She returned for a cameo appearance in the last episode of the second season, when she attempted to assassinate President David Palmer. Kirshner returned for the last episodes of the show's fourth season, in 2005.

She is also a main cast member of Showtime series The L Word, where she plays Jennifer Schecter, a sexually confused fiction writer.

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

In 2006, Kirshner starred in the third season of The L Word as Jenny Schecter. She will also star in Brian De Palma's upcoming movie The Black Dahlia, alongside Josh Hartnett, Aaron Eckhart, Scarlett Johansson and Hilary Swank. Kirshner will be playing the young actress Elizabeth Short, who was horrifically murdered in 1947.

She is also working on the book I Live Here. In the book, woman and children refugees from dangerous places such as Chechnya, Juarez (Mexico) and Africa will tell their disturbing life stories. It will be published in Canada in 2006.