The outcome of the Dutch referendum on the pact with Ukraine, seen as a barometer of anti-EU feeling, is viewed as an embarrassing blow both to the Netherlands - currently in charge of the rotating EU Council presidency - and the EU itself.
The Dutch news agency ANP said that with 99.8 per cent of the votes counted the 'No' camp had won the day with 61.1 per cent. Only 38 per cent voted in favour of the two-year-old treaty with Kyiv.
It was triggered by an internet petition begun by Eurosceptic activists that attracted some more than 400,000 signatures. Campaigners said the referendum on Wednesday was a chance for Dutch voters to express frustration at the EU, in particular what they see as its desire to expand despite democratic shortcomings.
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The Netherlands is now the only member state not to have ratified the EU-Ukraine association agreement, which had already been approved by the upper and lower houses of the Dutch Parliament.
It is estimated that 32.2 per cent electorate turned out, passing the 30 per cent threshold, meaning the ballot is valid and must be taken seriously by the coalition government of Prime Minister Mark Rutte.
The referendum is non-binding, but Rutte said the vote would be "considered" if turnout was over 30 per cent.
Diederik Samsom, leader of the Labour party, the junior partner the governing coalition, said, "We can't ratify the treaty in this fashion."
Ignoring a clear 'No' result could be risky for Rutte's already unpopular government - which has lost further ground over Europe's refugee debate - ahead of national elections scheduled for no later than March 2017.
MEP reaction to the vote was swift, with the Greens/EFA joint leader Rebecca Harms saying, "Ukrainian citizens and civil society have bravely pushed to reform their country, to achieve stable democracy and the rule of law for their country.
"The EU-Ukraine association agreement has given concrete form to the EU's support of this and contributes a lot to the necessary reform process in the country and helps to lead it to greater stability."
Her Dutch Greens colleague Bas Eickhout agreed, saying, "Anti-European sentiment among a proportion of the Dutch electorate should not be used as an excuse to deny Ukrainian citizens the possibility to develop their country to a democratic state with rights, laws and freedom."
The vote was being closely watched by Europe and Moscow, and could prove an important yardstick only months ahead of the UK's Brexit referendum in June.
According S&D group foreign affairs spokesperson Richard Howitt, many Dutch voters chose not to participate either because they disagreed with holding a referendum or deliberately wanted to suppress the turnout to avoid the minimum threshold being reached.
"British voters must learn the lesson that unless they take part, extremists can win," said the MEP, who was in The Hague for the vote.
Ukip leader Nigel Farage, who spoke at a rally for the 'No' side in Amsterdam on Monday, tweeted his support for the result, saying that he had spoken to the organisers of the petition that led to the Dutch referendum and invited them to the UK to discuss Brexit.
A spokesperson for campaign group Leave.EU, Brian Monteith, said: "This humiliating rejection of the Ukraine agreement demonstrates that people don't have to support the EU and its expansionist agenda to feel European."
Dutch far-right leader Geert Wilders said, "It looks like the Dutch people said 'No' to the European elite and 'No' to the treaty with Ukraine. This is the beginning of the end of the EU".
"I hope that later, both in the United Kingdom and elsewhere in Europe, other countries will follow," he said earlier
On Thursday, Ukrainian President Petro Poroshenko said that Ukraine will expand its cooperation with the EU, despite the Dutch rejection.
Poroshenko said that the organizers wanted to "challenge the power of the EU, not the association agreement with Ukraine."
He said, however, that the result was "an attack on the unity of Europe and the spread of European values."