Gardiner set off relatively slowly but gradually picked up the pace to power to victory ahead of his rivals with a season's best time of 43.85
Steven Gardiner of the Bahamas won gold in the men's 400m at the Olympic Stadium Thursday, running a carefully balanced race to shut out the Americans again in an event they once dominated.
Anthony Zambrano of Colombia, the silver medallist at the 2019 world championships in Doha and third-fastest this year, took the silver.
Kirani James of Grenada, the 2012 gold medallist and the silver medallist five years ago in Rio, grabbed bronze.
American Michael Cherry, who ran a personal best, finished a disappointing fourth ahead of his compatriot Michael Norman to continue the country's poor run on the men's side of the track events.
The US had won seven straight golds beginning in 1984 to 2008.
But they have failed to win the title since then, with James securing gold in 2012 and South Africa's Wayde Van Niekerk winning in Rio five years ago.
Thursday, a terrific run by Gardiner proved decisive.
He set off relatively slowly but gradually picked up the pace to power to victory ahead of his rivals with a season's best time of 43.85, adding the Olympic title to the world gold he won in Doha in 2019.