The 24-year-old raced home at the Golden Spike meet at the Mestsky Stadium in the northeastern Czech city of Ostrava in an outstanding 30.81 seconds.
The previous world record of 30.85sec was set at altitude in Pretoria in 2000 by American Michael Johnson.
It was the second time the 24-year-old South African has usurped Johnson, having broken his 17-year-old 400m world record when racing to victory at last year's Rio Olympics.
Van Niekerk's time also shattered the meet record of 30.97sec -- previously the second fastest time ever run -- set by Jamaican sprint star Bolt in 2010.
"I'm so pleased and grateful for this big achievement," said Van Niekerk, whose first professional appearance on the IAAF circuit was over 400m at Ostrava.
"Thanks to all the people of Ostrava for this wonderful meeting. I'm honoured to be a small part of history.
"I have to rest a little but after that I hope I can continue to make babysteps to be better day by day."
Running in lane five, Van Niekerk enjoyed a scintillating start that saw him catapult down the back stretch and pass Isaac Makwala of Botswana after the first 80 metres.
Negotiating the bend with aplomb, the South African shot into the home straight well in the lead.
"Thanks to all the people of Ostrava for this wonderful meeting. I'm honoured to be a small part of history.
"I have to rest a little but after that I hope I can continue to make babysteps to be better day by day."
Running in lane five, Van Niekerk enjoyed a scintillating start that saw him catapult down the back stretch and pass Isaac Makwala of Botswana after the first 80 metres.
Negotiating the bend with aplomb, the South African shot into the home straight well in the lead.
