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 last week.
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.
The 24-year-old timed a world lead of 43.62sec in Lausanne, well ahead of second-placed Botswana's Baboloki Thebe. 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.
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, Van Niekerk said he was constantly inspired by his performances.
"I didn't come with a specific time in mind but I am pleased with this result," he said.
"I do have a love-hate relationship with the 400m, but I always try to approach it with a positive mindset, not setting myself any limits.
"And the performances keep me going along, which keeps me passionate about what I do."
Gatlin, the 2004 Olympic 100m champion who has served two doping bans, clocked 9.96sec in the 100m, nipping in ahead of Ivorian Youssef ben Meite (9.98) and South African sprint hope Akani Simbine. (9.99).
"I didn't have a great start," said Gatlin, 35.
"I'm not at my A-game at the moment and that shows I'm a human.
"I am still a competitor. Now I will be able to go back to training knowing that my bases are are in place."
Dutchwoman Dafne Schippers timed 22.10sec, a seasonal best, to win the 200m from Ivorian Marie-Josee Ta Lou (10.16).
"I improved my best performance today and that makes me very happy," said Schippers, the 2015 world champion and silver medallist at the Rio Olympics last year.
"It's always nice to win. My objective this summer is to get a medal in London," where she will go for the sprint double, she said.
Ethiopian Genzebe Dibaba, the 1,500m world champion, raced to a world lead of 4:16.05 in the women's race over one mile.
"I felt good during the first part of the race. I had good legs up until 1200m. And then it blocked," she said.
"It's a pity as the conditions were good. I need to train some more to have some freshness at the end of the race."
American double Olympic champion Christian Taylor was trumped in the men's triple jump by Cuban Pedro Pichardo.
Pichardo managed a best of 17.60 metres, Taylor in second with 17.49.
American Sam Kendricks again got one up on Frenchman Renaud Lavillenie in the men's pole vault.
Kendricks, bronze medallist in Rio behind Lavillenie, cleared 5.93m, winning on countback from Poland's Pawel Wojciechowski with the French vaulter in third (5.87).