Michael Andrew of the US beat the London 2012 Olympic champion by touching first in a time of 51.96 seconds.
Le Clos had to be content with sharing the silver medal with Russian Egor Kuimov with both clocking 52 seconds.
Le Clos is looking to win his fifth Swimming World Cup overall series title which is held over seven legs and divided into three clusters.
Last year he became the first men’s swimmer to win four overall series titles adding to his 2011, 2013 and 2014 victories in the annual short-course series.
The first cluster consists of two galas in the 50m pool with the remaining races held over the 25-metreshort course.
Commonwealth Games bronze medallist Ryan Coetzee missed out on a medal, finishing seventh in the 50m freestyle with a time of 22.66sec.
Russia’s Vladimir Morozov won the ‘splash and dash’ event in a time of 21.49 with Andrew claiming silver in 21.99.
Le Clos has two more days to earn podium places with the South African superstar set to compete in his pet 200m butterfly event today.