A Dutch vote against strengthening ties between the European Union and Ukraine is an "attack on the unity of Europe," Ukrainian President Petro Poroshenko said Thursday, while EU officials insisted their approach towards Kiev would not change.

Dutch voters on Wednesday resoundingly rejected a referendum on the EU-Ukraine association agreement, meant to boost the 28-member bloc's political and commercial ties with the former Soviet republic.

"The true goal of the organizers of this referendum is not the association agreement between Ukraine and the EU. ... This is an attack on the spread of European values," Poroshenko said in a statement.

With all votes counted, 61 per cent opposed the referendum, while 38 voted in favour. Voter participation was 32 per cent - above the required minimum of 30 per cent for the vote to be valid.

The vote was non-binding, but many see the rejection as an embarrassment to the Netherlands - which holds the EU's rotating presidency until the end of June - and something that the Dutch government will have little choice but to act on.

EU lawmaker Manfred Weber criticized Dutch Prime Minister Mark Rutte and the country's elites for not being present enough ahead of the referendum. He said the result was "anti-Rutte, anti-Europe; it was anti-migration, anti-everything," in an interview with dpa.

Criticism also came from Moscow, which has been at loggerheads with Kiev since pro-EU protests led to the downfall in 2013 of former Ukrainian President Viktor Yanukovych, who had previously backed away from the association deal with Brussels.

"The results of the Dutch referendum on the EU-Ukraine association agreement indicate Europeans' opinion of the Ukrainian political system," Russian Prime Minister Dmitry Medvedev wrote on Twitter.

Kremlin spokesman Dmitry Peskov said the Dutch No vote exemplified European mistrust of the Ukrainian government.

The political and free trade deal has been signed by both sides and applied provisionally, pending full ratification. All of the EU's member states, except the Netherlands, have given their final approval.

EU President Donald Tusk emphasized on Thursday that the agreement remains valid.

"I have taken note of the reported outcome of the referendum in the Netherlands. I will continue to be in contact with Prime Minister Rutte on this, as I need to hear what conclusions he and his government will draw from the referendum and what his intentions will be," Tusk said.

German Chancellor Angela Merkel said Ukraine should remain on an "uninterrupted" path, while French President Francois Hollande said Berlin and Paris wanted to continue their support for Ukraine. They were speaking during a Franco-German summit in the French city of Metz.

But German Foreign Minister Steinmeier said the result was "surely no good day for Europe."

British Prime Minister David Cameron meanwhile was hoping that the Dutch decision will not influence his country's referendum on a so-called Brexit.

The British referendum was on "a very different issue," Cameron said talking to students in Exeter on Thursday.

Britain will vote on June 23 whether or not to stay in the EU.

European Commission President Jean-Claude Juncker was "sad" at the Dutch referendum outcome, his spokesman Margaritis Schinas said.

Juncker had warned earlier this year that a No vote could "open the door to a major continental crisis."

"The commission remains strongly committed to the development of its relations with Ukraine," Schinas said, adding that the EU's executive would later this month present a proposal to grant Ukrainian citizens visa-free travel, as planned.

Poroshenko also said that Ukraine did not consider its cooperation with the EU to be in danger.

"I am confident that this event is not a strategic obstacle for Ukraine on the path to Europe," Poroshenko said. "We will not turn off the road of European integration."

The United States also expressed disappointment in the vote's outcome and said a closer relationship for Ukraine with the West is in the country's best interest.

"Clearly we're disappointed by the results, but we do respect the views of the Dutch people and we respect the Dutch political process," State Department spokesman Mark Toner said.

Until the association agreement is fully ratified, however, not all chapters apply.

Sections that are suspended for the time being cover issues such as foreign and security policy, conflict prevention, arms control and the fight on terrorism.

Chapters on domestic issues such as asylum and border management, money laundering, the fight on drugs, crime and corruption and judicial cooperation are also currently not being applied.

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