Sixty-four percent of voters rejected a far-reaching free trade deal with Ukraine, but an exit poll found that low turnout may have invalidated the referendum.
Voters were asked whether they would support the association agreement with Ukraine, which aims to open the war-torn country and former Soviet satellite up for EU trade. The exit poll had a margin of error of at least 3 percent.
"It is clear that 'no' have won by an overwhelming margin," said Prime Minister Mark Rutte, who had voted to approve the referendum. "If the turnout is above 30 percent with such a large margin of victory for the no camp," he added, "then my sense is that ratification can't simply go ahead."
It remains unclear whether turnout did reach the required 30 percent. Exit polls initially put turnout at 29 percent, before updating it to 32 percent with a margin of error of 3 percent.
'A continental crisis'
The other 27 EU member states have provisionally approved the agreement, which has gone into effect, as had both houses in the Dutch parliament.
The "no" campaign highlighted Ukraine's corruption and unrest after a 2014 popular uprising ousted Moscow-backed President Viktor Yanukovych, who had rejected the cooperation deal.
Current President Petro Poroshenko had expressed optimism ahead of the vote, but perhaps saw his credibility damaged after this weekend's release of the "Panama Papers," in which he was named.
A valid "nee" could pose a new headache for the EU. Ahead of the vote, European Commission chief Jean-Claude Juncker warned that a no vote "could open the doors to a continental crisis."
Political observers in the Netherlands have said that Wednesday's poll is being used as a gauge of anti-EU sentiment ahead of a June 23 UK vote on whether to remain in the 28-member bloc. Far-right leaders openly expressed optimism that a win for the "no" campaign would boost the backers of Britain's exit, or "Brexit."
"If the Dutch people vote no today, it will be a incentive for the British voters to say no," said Geert Wilders, a member of parliament and the leader of the anti-immigrant Freedom party. "So," he added, "it could be today that it is the start of the end of the European Union as we know it today - and that would be very good."
The Netherlands currently holds the rotating EU presidency.
mkg/jm (Reuters/AFP, dpa, AP)