A Dutch referendum is to be held next Wednesday on an EU agreement for closer relations with Ukraine after 427,000 people backed a citizens' initiative.
A new Dutch law gives any citizen the right to request a non-binding referendum on any law that has been voted on by Dutch Parliament but which hasn't become active yet.
The Dutch Parliament had already backed the association agreement, which removes trade barriers between the EU and Ukraine, and some have argued that the outcome will not make any difference to the agreement.
Even so, European Commission President Jean-Claude Juncker has warned of "major consequences" if the agreement fails.
He has urged Dutch voters not to oppose the deal with Ukraine, saying such a move "could open the doors to a continental crisis."
He told NRC, a Dutch newspaper, "Let's not change the referendum into a vote about Europe."
Although the result of the poll on 6 April will not be binding on the government, it will have to be considered if turnout is above 30 per cent and a majority votes against the agreement.
A founding member of the EU, the Netherlands has in recent years cooled on European integration amid controversies about immigration from poorer new members in Eastern Europe.
The Dutch coalition Liberal-Labour government of PM Mark Rutte decided not to actively campaign for a "yes" vote but has said it will examine the results.
Campaigners in the "Vote No" camp fear that it could be a step towards EU membership and would cost Dutch taxpayers billions of euros.
The "No" side argues that Dutch voters should have their say on policies such as EU expansion, legislation and aid packages and is supported by the populist right-wing MP Geert Wilders.
Although the result will not be binding on the government, it will have to be considered if turnout is above 30 per cent and a majority votes against the agreement.
Kateryna Zarembo, a deputy director of the Institute of World Policy, a think-tank in Kiev, Ukraine, said, "Some argue that vote is not really about Ukraine - it's about distrust of national politics and the country's growing Eurosceptic sentiment."
"A No vote would deal a serious blow to the EU, just as the world around us becomes more unstable and uncertain - and thus requires more, not less EU involvement. If the Dutch vote Yes, EU citizens would have as many reasons to celebrate the outcome as would the Ukrainians."
UKIP leader Nigel Farage has also waded into the debate, saying, "This push for a Dutch referendum shows that many peoples across Europe, not just the British, are tired of the EU forcing laws, expenses and migrants onto them without their consent."
"This is now the Dutch peoples' chance to show the Brussels bureaucrats that national democracy cannot be ignored forever."