Canadians Denny Morrison, Lucas Makowsky and Mathieu Giroux won the eight-lap, 3,200m race with a time of three minutes 41.37 seconds.
Canadian coach Marcel Lacroix pounded the ice as his skaters crossed the line .21 of a second ahead of the Americans at Richmond Olympic Oval.
"It's a weight off the shoulders," Morrison said. "It's a race where you represent Canada.
"I cannot even think what it means. I was not happy with the results (in the individual races) but I knew that it could work in the team pursuit. We had what it took."
The crowd erupted in cheers with Canadian Prime Minster Stephen Harper and double short track gold medallist Charles Hamelin in the stands joining the thunderous reaction.
The US team, comprised of 1000m bronze medallist Chad Hedrick, Brian Hansen and Jonathan Kuck, took the silver.
The Netherlands, with 5000m champion Sven Kramer, 1,00m winner Mark Tuitert and Jan Blokhuijsen, took bronze by beating Norway by 0.55 seconds.
"It was pretty good for today, but I am a little bit disappointed from yesterday because we know we are one of the best teams in the world," Kramer said.
The Canadian iron men, who skated all three rounds of the event without mixing in a fourth skater as all the teams had, climbed the podium and jumped for joy together after finally claiming their Vancouver medal in the last men's event.
Makowsky said: "We won gold and it's one heck of a way to finish the Games."