Welsh Conservative leader Andrew RT Davies has said he will be voting for Britain to leave the EU in June.
"Our future will be best served as part of a looser, economic relationship with the European Union," he said.
Meanwhile, the prime minister has warned that leaving the EU would mean poorer parts of Wales would lose out on European grants.
Speaking in the House of Commons, David Cameron said EU funds had "made a big difference" in parts of Wales.
However, Mr Davies said that before the EU referendum, voters in Wales faced "an even bigger question" before then at the assembly election in May.
The Tories would "not be distracted from articulating our positive vision of real change for our NHS, education and economy," he said.
"After a great deal of consideration I have decided that I will be voting for Britain to leave the EU. It is my belief that our future will be best served as part of a looser, economic relationship with the European Union," Mr Davies said in a statement on Monday.
"There will be almost seven weeks for Welsh politicians to articulate their positions on the European question, but before a ballot has been cast in the referendum Welsh voters face an even bigger question.
"We are a little over 70 days away from the most important Welsh assembly election in a generation and the Welsh Conservatives will not be distracted from articulating our positive vision of real change for our NHS, education and economy."
Analysis by Nick Servini, BBC Wales political editor
So, Andrew RT Davies has "done a Boris".
This has potentially big implications for the assembly campaign because I find it hard to believe that Labour, and the other parties for that matter, will not try to use it against him.
Carwyn Jones has already portrayed the assembly contest as coming down to a straight question of who you want as first minister: him or Andrew RT Davies.
Now it is surely a matter of time before an opponent says he should not be first minister because he wants to take us out of the EU.
Whatever the merits of that criticism, the point is the EU debate will then become a central part of the assembly debate as well.
Jobs 'at risk'
Welsh Liberal Democrat leader Kirsty Williams said she was "staggered" by Mr Davies's support for a UK exit from the EU.
"His vote to leave is an affront to the hard-working farmers, small business owners and other people whose jobs depend on EU trade," she said.
Labour's Shadow Welsh Secretary Nia Griffith claimed Mr Davies was "happy" to put 200,000 Welsh jobs "at risk".
"There's now a clear choice for first minister in May: Labour's Carwyn Jones and his record of delivery, or a Tory leader prepared to gamble with Welsh jobs," she added.
The Welsh Tory leader's declaration puts him at odds with the prime minister and with Welsh Secretary Stephen Crabb, who said supporters of a UK exit from the EU were offering "confusion and vagueness".
Of the 11 Welsh Tory MPs, six have said Britain should remain, four want to leave and one was undecided.
Mr Crabb said remaining in the EU was right for Welsh businesses and claimed the "overwhelming majority" of firms agreed.
"There's a lot they don't like about the European system in the same way that there's lots they don't like about Welsh government and UK government," he said.
"But they know that on a balance of costs and benefits, risks and opportunities, Britain and Wales are actually better off staying part of a reforming European Union."
Mr Crabb's junior Wales Office minister Alun Cairns was the sixth Welsh Conservative MP to confirm he wanted the UK to remain in the EU.
Earlier on Monday, Vale of Clwyd Tory MP Dr James Davies declared on his Facebook page he wanted the UK to leave the EU, saying it was "time to move on".
Montgomeryshire MP Glyn Davies revealed on Twitter he would seek the views of local party members before deciding which way to vote.
Labour, Plaid Cymru and the Liberal Democrats have said their parties will campaign to keep the UK in the EU, while UKIP will campaign to leave.
Meanwhile, speaking in the Commons on Monday, David Cameron said EU funds had "made a big difference" in parts of Wales.
He was responding to a question from Plaid Cymru's parliamentary leader, Hywel Williams, who asked, if Britain voted to leave: "What would happen to convergence funding which has provided large amounts of money for the poorer areas of west Wales and the valleys?"
Mr Cameron told him: "If we were to leave the EU then we wouldn't be able to get those funds which have made a big difference in parts of Wales, parts of England, for instance in Cornwall and in other parts of our country."