The move by New Democratic Party (NDP) Member of Parliament Claude Patry will not have an immediate effect on the balance of power, but it is an important boost for the separatist cause after its poor showing in the 2011 federal election.
It is also embarrassing for the NDP - a federal party dedicated to keeping Canada intact - which became the official opposition for the first time following the 2011 election.
Patry said he wanted Quebec to become an independent.
"Like many Quebec citizens in 2011, I thought the NDP would act differently than the Liberals and Conservatives and that it would truly recognize the aspirations of the Quebec nation," he said in a statement.
Quebec voters abandoned the Bloc in droves in 2011 in favor of the center-left NDP, which struck a careful balance between defending the province's role inside Canada while making clear it should not secede.
Patry's defection means the NDP now has 100 legislators in the House of Commons, 57 of whom are from Quebec, while the Bloc has just five.
Prime Minister Stephen Harper said Patry's defection reflected what he called a worrying ambiguity inside the NDP parliamentary caucus about Canadian unity.
The ruling Conservatives have 165 of the 308 seats in the House of Commons. The next election is not due until late 2015.
Quebec's relationship with Canada is one of the country's most delicate political issues. The province only just failed to vote for independence in a 1995 referendum, prompting the Liberal federal government at the time to pass the Clarity Act - which says Quebec can only separate with a clear majority on a clear question.
The NDP last month proposed replacing the Clarity Act with a law which would let Quebec secede if a simple majority voted in favor of a simple question. This angered separatists, who say Quebec alone should decide referendum questions.
NDP leader Thomas Mulcair said Patry had agreed to the party's positions on Quebec before becoming a candidate and said the legislator should resign and try to win back his seat as a member of the Bloc. Patry would fail, he predicted.