Exit polls showed about 62 percent of voters said no to the treaty, on a turnout of around 64 percent.
The results in the Netherlands, one of the EU’s founding members, exposed deep divisions over the direction of the bloc.
Conceding defeat Prime Minister Jan Peter Balkenende admitted he was “disappointed” by the result but promised to honour the outcome.
“It is a clear outcome. Of course I am very disappointed,” he said.
The stinging defeat comes three days after France also voted the it down.
The treaty aims to streamline decision-making in the bloc following its historic enlargement last year, when 10 mostly ex-communist countries joined.
For the constitution to be adopted, it needs to be ratified by all 25 member states. Nine countries have so far approved it.
Mr Balkenende said the process of ratifying the treaty should continue in the rest of Europe “to know where each country stands”.
His call was echoed by the EU’s Luxembourg presidency and German Chancellor Gerhard Schroeder.
In contrast to the shocked mood in much of Europe, Dutch far right
legislators were jubilant after their vigorous “no” campaign.
“I had not expected this massive turnout. I am proud of the Dutch people,” said populist Geert Wilders, who defected from the Liberals to form a far-right party with one seat in parliament.
Analysts say the Dutch “no” vote was not a rejection of European integration but a warning about its pace, coupled with a sense of disillusionment with politics in general.
“I voted no because I don’t trust the government,” said one elderly Amsterdam resident, who said he was unhappy about EU enlargement last year.
Surveys show the Dutch fear a rapidly expanding EU could swallow up their nation and that focusing power in Brussels could eventually force the Dutch to revise liberal laws on cannabis, same-sex marriages and euthanasia.
“In the Netherlands, more than 40 percent of the people think that Europe is moving too fast with the euro and enlargement with eastern European countries followed by Turkey,” said Maurice de Hond, director of a polling institute.
European Commission chief Jose Manuel Barroso warned any countries thinking of abandoning the constitution not to be hasty ahead of a June 16-17 European Union summit in Brussels.