American Shani Davis, meanwhile, failed to generate his typical power and finished eighth. He came up far short in his bid to become the first man from the United States to win the same event at the Winter Olympics three times in a row, something snowboarder Shaun White was also unable to do 24 hours earlier.
Groothuis, 32, who has admitted to seeking medical help for bouts of severe depression, finished fourth in the 1,000-meter race at the Vancouver Olympics. He was not among the favorites for gold since the winner and runner-up from four years ago, Davis and Tae Bum Mo of South Korea, were in the race.
The opening 200-meter time for Groothuis was only the fourth fastest of the day, but he powered his way through the remainder of the race to turn in a time of 1:08.39.
Eight skaters were left to compete, including Davis and Mo, but the closest anybody could come to Groothuis was Canada's Denny Morrison.
Morrison won the silver medal in 1:08.43 and Mulder finished third in 1:08.74. Mulder won the 500-meter gold medal earlier in these Olympics.
It was the latest chapter in what has been a stunning performance by the Netherlands at the speed skating oval.
This was the fifth race at the Sochi Games and skaters from the Netherlands have won four of them. In addition, that country has collected 10 of the 15 medals awarded in speed skating. The Netherlands has not won a medal in any other sport at the 2014 Olympics.
That haul has included a sweep of the medals in the men's 5,000-meter race, where Sven Kramer won the gold. Kramer will be favored to win the 10,000-meter event as well and the Netherlands will be expected to capture the men's team pursuit race.
The showing by Davis was a shock to those who had gathered expecting to see a big show by the two-time defending champion. He has had three victories this season on the World Cup circuit.
Davis skated the first 200 meters just 0.02 second off the time Groothuis turned in, but the drive in his legs seemed to melt away during the middle portion of his race and he could not even finish in front of the skater with whom he was paired -- Koen Verweij of the Netherlands.