The 22-year-old University of Free State sprinter clocked 44.24 seconds to win his specialist discipline at the Diamond League meeting in New York.
Van Niekerk improved his own national mark of 44.38, set at the same meeting last season, to bag the second Diamond League victory of his career.
He crossed the line 0.50 ahead of five-time World Indoor Championships medallist Christopher Brown of the Bahamas, who took second place.
“I just took it step by step and tried to stay comfortable,” Van Niekerk said. “I am happy with the result and I’m looking forward to the rest of the season.”
In the men’s 400m hurdles race, former Commonwealth Games champion LJ van Zyl produced his best performance in more than three years.
Van Zyl finished second behind Javier Culson of Puerto Rico, covering the one-lap distance over the barriers in 48.78.
Akani Simbine ended third in the men’s 100m dash in 10.18, running into a headwind of 1.7 metres per second.
Keston Bledman of Trinidad and Tobago finished ahead of the South African, but he was disqualified for a false start after running under protest, as Simbine secured his first podium place at a Diamond League meeting.
Controversial American Tyson Gay, a former world champion, won the tightly contested race in 10.12. Pretoria giant Orazio Cremona ended eighth with another relatively disappointing performance in the men’s shot put.
Cremona launched a 19.51m heave, with his best attempt falling more than two metres short of American winner Joe Kovacs.
There is now a three-week break in the top-flight Diamond League series. The next leg of the campaign will be held in Paris on July 4.