Unofficial final results indicated 61.6 percent of Netherlands voters said no to the proposal.
The BBC said Netherlands Prime Minister Jan Peter Balkenende, who campaigned for a yes vote, said he would respect the result, but admitted he was "very disappointed."
The Dutch voter turnout was 62.8 percent, which was more than double what was needed for the vote to be accepted as the public's verdict on the treaty.
Since some absentee ballots were still to be counted, the official result is not expected till June 6.
"I think there's a revolt against the establishment that leaves governments from Great Britain to France to Germany to Italy singularly weak," Charles Kupchan, as associate professor of international relations at Georgetown University, told the New York Times.
A fellow at the Council on Foreign Relations, Kupchan added, "That spells trouble for Europe and it spells trouble for an America that will be looking to Europe for help on many different fronts."
Nine nations have ratified the constitution, including Germany. But it must be approved by all of the EU's 25 member states to become law.