The South African's time of 48.08sec improved his own world record of 48.44, set at the previous edition of the championships in Doha in 2014.
Le Clos, denied a 200m fly Olympic repeat in Rio, added another gold to the 200m fly title he captured on Tuesday when the championships kicked off in Windsor, Canada.
American Tom Shields was second in 49.04 and Australian David Morgan claimed bronze in 49.31.
Hungary's one-woman wrecking crew of Katinka Hosszu, a treble gold medallist in Rio, picked up her fourth individual gold of the championships in the 200m backstroke.
It was another convincing win for the "Iron Lady," who was in control from the 50m mark and steadily built her lead to win in 2:00.79 -- more than a second in front of runner-up Daryna Zevina of Ukraine (2:02.24). Australian Emily Seebohm took bronze in 2:02.65.
Hosszu, winner of the 200m butterfly, 100m back and 400m individual medley earlier in the meeting, also topped the semi-final times in the 100m individual medley.
But she wasn't a factor as she capped her night with an eighth-place finish in the 800m freestyle won by American Leah Smith.
Smith, the 400m free bronze medallist in Rio, led all the way to head a US one-two in 8:10.17. Ashley Twichell was second in 8:11.95 and Australian Kiah Melverton was earned bronze in 8:16.51.
The 800 free field was missing reigning champion and world record-holder Mireia Belmonte of Spain, who pulled out of the event after a disappointing showing in the 400m medley on Tuesday.
Koch snags second gold
Germany's Marco Koch further soothed the disappointment of Rio with a victory in the 200m breaststroke, adding a second gold to the 100m breast title he captured on Wednesday.
Koch, the reigning long course world champion who was relegated to seventh in the 200m breast at the Olympics, clocked a championship record of 2:01.21 for a comfortable win over Britain's Andrew Willis (2:02.71) with Russia's Mikhail Dorinov third in 2:03.09.
Australian Brittany Elmslie, a relay gold medallist in Rio, snatched victory in the women's 100m freestyle, earning her first short course world title in 51.81.
Silver went to 2012 double Olympic sprint gold medallist Ranomi Kromowidjojo of the Netherlands, who was second in 51.92, while Canadian teenager Penny Oleksiak, who shared 100m free gold with American Simone Manuel in Rio, took bronze in 52.01.
The United States capped the night with a victory in the mixed 4x50m medley relay. Michael Chadwick, swimming the final freestyle leg, hit the water in seventh place but stormed home to complete the victory in 1:37.22.
The win gave breaststroker Lilly King her third gold of the championships, after an individual triumph in the 50m breast and her swim on America's world record-setting women's 4x50m medley relay.