The outcome has been claimed as a victory by Out campaigners in the UK.
With a turnout of near 32 per cent, 61 per cent voted "no", while 28 per cent supported the deal.
The vote is non-binding, so a "no" vote has not forced the government into a veto, but puts Prime Minister Mark Rutte in a difficult position, especially as his government currently holds the EU's rotating presidency.
However, Rutte said: "My view is that if the turnout is more than 30 per cent, with such a victory for the "no" camp, ratification cannot go ahead without discussion."
Pro-Brexit campaigners said the result would spur on the Leave campaign in the UK. Leader of Ukip Nigel Farage said the result will strengethen the Leave campaign.
"This result gives the British people the signal that it is moderate and normal to reject the EU and stand up for what's in our country's best interests. The sun is now setting on the European Union," said Leave.EU spokesman Brian Monteith.
However, pro-Remain campaigners, as well as Professor John Curtice, president of the British Polling Council, previously questioned if the result would have any impact on the UK's own vote.