Prime Minister Zoran Milanovic nevertheless welcomed the result.
"Croatia decided that it will be a member of the European Union," he said.
"It is a historic decision ... possibly a turning point in our history."
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The provisional results issued late Sunday were based on votes counted at almost 99 percent of polling stations.
Only a simple majority was required and the vote is valid regardless of turnout.
But one anti-EU group nevertheless argued that the results were not valid precisely because of the 43.58 percent turnout announced by the election commission.
That figure was substantially lower than the 54.32 percent turnout for December's general election, which Milanovic attributed to general disillusionment with politicians and the country's economic difficulties.
"People are obviously disappointed...," said Milanovic, who only took office a month ago.
"This is a message due to the situation in the country, a message to my government," said Milanovic, whose centre-left coalition led by his Social Democrats (SDP) ousted the scandal-plagued conservative HDZ.
For President Ivo Josipovic however, "most important is that the big European 'yes' has been said."
But the authorities should also take into account fears and dilemmas of those who voted 'no,' he added.
All major Croatian political parties are in favour of EU membership.
They see it as a vital factor for consolidating peace and economic recovery in the ex-Yugoslav republic, which gained independence in 1995 after a four-year war with rebel Serbs.
For the past three years Croatia, whose economy relies on Adriatic tourism, has been struggling economically. The central bank is forecasting that the economy will shrink 0.2 percent this year.
But as well as its economic problems, the country has had to deal with a series of top-level corruption scandals.
Although they mostly involved the former ruling conservative HDZ party, many observers feel that the fall-out has been a general mistrust of politicians.
After the results were announced, one spokesman for the anti-EU camp threatened legal moves arguing that the low turnout invalidated the referendum.
"The Croatian people have turned their backs to local political leaders," Zeljko Sacic, of the 'Council for Croatia - No to EU' umbrella group, told national television.
"This is a defeat of Croatia's freedom (and) independence ... We are entering an association that is falling apart," he warned.
But legal specialists say legal challenges to the validity of the referendum have little chance of succeeding: the law was changed in June 2010 precisely to remove the minimum turnout requirement.
Sacic's comments nevertheless represented a popular view among the sceptics, who do not think the EU has much to offer given it is battling its own debt crisis.
Those opposed to entering the European Union have also expressed fears about a loss of sovereignty and national identity in this country of 4.2 million.
While other post-communist countries in central and eastern Europe were strengthening their democracies and paving their way towards EU integration, Croatia's EU aspirations were halted by the 1991-95 war and its legacy.
It was not until 2000 that the election of a pro-European government enabled Croatia's transformation into a genuine parliamentary democracy eligible for EU candidate status.
However, enthusiasm for EU membership waned after long and often difficult accession talks that opened in 2005.
Croatia's EU accession treaty would still have to be ratified by all 27 members of the bloc, before the country formally joins on July 1, 2013.
Slovenia is the only current EU member among the six former Yugoslav republics, though Serbia, Montenegro, Macedonia and Bosnia all have aspirations to join.

