Negin, most prominently a stage actor, had his earliest film and television roles in the 1950s Canadian dramatic anthology series First Performance, and as a chorus member in Tyrone Guthrie's 1957 film of the Stratford Festival production of Oedipus Rex. He appeared in the Stratford Festival production of Tamburlaine, which had a run on Broadway in 1956, and later appeared in London productions of Fortune and Men's Eyes and his own play Love and Maple Syrup; in Fortune and Men's Eyes, he became one of the first actors ever to appear fully nude on stage in England.
He later appeared in films including The Ernie Game (1967), Can Heironymus Merkin Ever Forget Mercy Humppe and Find True Happiness? (1969), Ooh… You Are Awful (1972), Barry McKenzie Holds His Own (1974), Rabid (1977), Two Solitudes (1978) and Highpoint (1982), as well as TV series such as Brett, Mousey and The Zoo Gang and episodes of King of Kensington and The Littlest Hobo. In the 1980s he had a recurring role on Seeing Things, as well as acting in the television films Overdrawn at the Memory Bank, Freddie the Freeloader's Christmas Dinner and Charlie Grant's War.
In 1994, he acted in drag as Mrs. White in a Toronto production of John Wimbs and Christopher Richards' play Molly Wood. In 1998, he played Noël Coward in a production of Linda Griffiths' play The Duchess at Theatre Passe Muraille. He played Truman Capote, both in a Toronto stage production of the play Tru in 1996 and in the film 54.
In recent years, he has acted in several of Guy Maddin's films, including Cowards Bend the Knee, Sissy Boy Slap Party, The Saddest Music in the World, Keyhole, and The Forbidden Room as well as narrating Maddin's semi-documentary films Brand Upon the Brain! and My Winnipeg. He has also had guest roles in the television series Lord Have Mercy!, Mona the Vampire, ReGenesis and Slings and Arrows, and in Bruce McDonald's film Pontypool.
Negin, who is gay, is the partner of Canadian former television and film designer Charles Dunlop. In a 2007 interview with Xtra!, Negin described his age as "Write that I'm 95 years old, and that I've been to Hungary to have some work done."
- "Enchantment". In Toronto, September 2011.
- Louis Negin at the Canadian Theatre Encyclopedia.
- "Molly Wood goes the way of all flash". Toronto Star, October 27, 1994.
- "Toronto Tru Delays Opening" Archived 2012-10-22 at the Wayback Machine. Playbill, May 23, 2996.
- "Silver screen: Louis Negin's shocking disclosures". Xtra!, May 24, 2007.