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The world and Olympic champion, also the world record holder in the event, hared off down the back stretch in lane five and looked dead set for another straightforward victory.
But Makwala, in the lane outside, battled back around the final bend into the home strait.
Van Niekerk, however, dug deep and surged home in a meeting record of 43.73sec, Makwala setting a personal best of 43.84 in second.
“It was a great performance today,” said the South African.
“I’m feeling positive about it. My body feels to be in great shape and this win from behind gives me confidence.
“We still are not peaking, we trained hard last week and all should be okay for London and my double (200, 400m).
“I’m ready for the big plan!”
Van Niekerk is on fire this season, having run personal bests in the 100m (9.94sec) and 200m (19.84) last month, and setting a new world best time of 30.81sec in the rarely-run 300m in Ostrava earlier this month.
That time beat a long-time record held by Michael Johnson, the legendary US athlete whose record in the one-lap race he also beat when winning gold in Rio.
In his last Diamond League outing, after Ostrava, the 24-year-old timed a world lead of 43.62sec in Lausanne.
Van Niekerk has only run the 400m faster on two occasions: when he won his world title in Beijing in 2015; the other was a year later when he won Olympic gold.
It is no surprise the South African has been hailed by eight-time Olympic champion Usain Bolt as his successor as leader of the sprinting world, with the Jamaican legend due to retire after the world championships in London in August.
Makwala will be racing Van Niekerk over six solid days at the August 4-13 worlds, after admitting he would also double up.
“The race felt good from start to finish! I have now decided I will be doubling the 200-400m in London,” the Botswanan said.
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