Canada shocked the field by earning an apparent bronze medal on Saturday before being disqualified moments later.
The Jamaicans, meanwhile, broke their own world record en route to a gold medal.
The disqualification came when Jared Connaughton stepped on the line of his outside lane just prior to his exchange with Justyn Warner, Canada’s anchor.
A distraught Connaughton, of New Haven, P.E.I., knew immediately that he had committed the stringent infraction and apologized to the rest of his teammates — lead Gavin Smellie (Brampton, Ont.), Oluseyi Smith (Ottawa), and Warner (Markham, Ont.).
He later tweeted: "I'm so sorry everyone. My heart is broken. I let my team down. I'm sorry."
Athletics Canada appealed the disqualification, but the decision to keep Canada off the medal podium was upheld.
“I take full responsibility,” said Connaughton while also calling the rule "stupid."
“I’m captain of this team and these guys are like my little brothers”
Jamaica, comprised of Nesta Carter, Michael Frater, Yohan Blake, Usain Bolt, set the new world mark in 36.84 seconds.
The Americans, a team consisting of Trell Kimmons, Justin Gatlin, Tyson Gay and Ryan Bailey, finished second with a time of 37.04, which matched the previous world mark set by Jamaica. Trinidad and Tobago replaced Canada as the bronze medallist at 38.12.
"It's always a beautiful feeling to end off like this," Bolt said. "We did it last year in the world championships — for me it is a wonderful feeling."
Canadians held firm
Even with Great Britain — the world’s No. 4-ranked team — out of the final after a disqualification for an illegal exchange in the opening heat on Friday, Canada was in tough against a strong field.
But the 11th-ranked Canadians held firm as they remained in contention throughout the race. When Connaughton handed the baton to Warner for the final leg, Canada stood fifth.
Warner, who's been a revelation during these Olympics, quickly made up the ground and just edged out Trinidad and Tobago’s Richard Thompson at the line.
The Canadians were justifiably elated as they saw their result flash across the scoreboard, which displayed Canada in the No. 3 position. It appeared the nation would reach the 4x100 relay podium for the first time since Donovan Bailey led a gold-medal victory at the 1996 Atlanta Games.
However, celebration was quickly replaced with heartbreak when the Canadian members looked up at the same scoreboard and saw the team had been disqualified.
"We ran a great race and we did everything," Warner said. "This is unfortunate. It sucks. Everyone dreams about getting a medal and we have just had one taken away. It's not any of our faults and we are proud of what we did. We had the job done."
Jamaicans defend Olympic title
Jamaica, featuring 100 and 200 gold medallist Bolt, came into the competition as the defending Olympic champions and reigning world-record holders.
The U.S., which had 100 bronze medallist Gatlin on its side, would be the only country in the field capable of unseating Jamaica — on paper that is.
With Blake, the 100 and 200 Olympic runner up, racing the third leg, he immediately erased the American advantage. By the time Blake made the exchange with Bolt the race was essentially over.
Bolt blew past Bailey and headed toward the finish.
"He was basically the difference in the race. It was even all the way around," Gay said of Bolt. "When he got the stick, there was nothing we could do about it."
There would be no showboating on this occasion. Bolt never broke stride as he crossed the line — 0.20 seconds faster than the same quartet of sprinters who set the world record at last year’s world championships.
The average time of the four Jamaicans was a blistering 9.21 seconds.
It’s was another historic day for Bolt, who is now a perfect 6-for-6 in his Olympic racing career.
In Beijing four years ago, he completed a sweep of the 100, 200 and 4x100 events — all in world-record time.
On Saturday he finished off the same trifecta, with only the relay team breaking a world record.
Bolt becomes the only male sprinter to ever win gold in those three events at consecutive Olympics.