Van Niekerk, who broke Johnson's 400 metres world record at the Rio Olympics last year, pulled away from the pack around the curve on his way to a time of 30.81 seconds, beating the American Johnson's mark of 30.85 seconds set in 2000.
He also beat the Ostrava meeting record over the rarely-run distance set by multiple Olympic and world championship gold medallist Usain Bolt in 2010.
"I have been improving each in every one of my events so it is a great step for the year and I am feeling quite confident for the rest of the year," the sprinter, who is expected to run the 200m and 400m at the world championships in London in August, told Czech Television.
At the Rio Games last summer, Van Niekerk won the men's 400 metres gold in a record time of 43.03 seconds, bettering Johnson's previous mark of 43.18 seconds set in 1999. (Writing by Jason Hovet; Editing by Toby Davis)