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.
