Le Clos touched the wall in 48.44 seconds to chop 0.04 off the previous global mark set by Russian Evgeny Korotyshkin in Berlin in November 2009.
UPDATE: Chad le Clos bags fourth gold
Meanwhile, it was a night of near misses for the majority of the South African swimmers in action in the evening session on day two.
The national 4x200m freestyle relay team of Le Clos, Myles Brown, Sebastien Rousseau and Leith Shankland lost out in a tight contest, taking fourth place in the final in 6:52.13.
They chopped nearly six seconds off the SA record set in the morning session, but ended 0.17 shy of the bronze medal.
Cameron van der Burgh, the world record holder, settled for fourth position in the 100m breaststroke final, clocking 56.80 in only his third race since returning from a shoulder injury.
He was just 0.02 behind French bronze medallist Giacomo Perez-Dortona.
Rousseau also ended fourth in the 400m individual medley, in 4:02.00, trailing third-placed David Verraszto by 0.18.
The SA quartet of Ricky Ellis, Giulio Zorzi, Clayton Jimmie and Luke Pendock took eighth place in the men’s 4x50m medley relay final in 1:35.05.