The prime minister has told colleagues at the current G7 summit in Bavaria that all ministers will have to toe the official line.
Mr Cameron said that unlike Harold Wilson, who allowed Labour ministers to campaign on both sides of a Common Market referendum in 1975, he expected "everyone in government" to fight on the same side.
"I've been very clear that if you want to be part of the government, you have to take the view that we are engaged in an exercise of renegotiation, have a referendum and that will lead to a successful outcome," he said.
Meanwhile, Philip Hammond, the foreign secretary, dismissed demands by some Tory MPs in the new group for the Westminster parliament to have a unilateral veto over any EU law.
"That's not achievable, that's not negotiable, because that would effectively be the end of the European Union," he told the BBC. He said a group of national parliaments working together should be able to show a "red card".
However, the Tory MP Steve Baker, head of Conservatives for Britain, said it was a "modest demand that parliament should have sovereignty over its own territory". If that were not granted, Britain would "have to leave".
Read MoreBank of England mis-sends Brexit email to press
Mr Cameron will have his first clash with the new group this month when MPs debate amendments to the EU referendum bill, which paves the way for a British in-out vote by the end of 2017.
Tory eurosceptics could rebel against Mr Cameron for the first time since his May 7 election victory, over a provision that would allow the use of public money in the run-up to the vote to make the case for British EU membership.
The MPs want to see the normal 28-day "purdah" period of official neutrality before a sensitive vote. But Mr Hammond said the government had no intention of remaining "neutral" in the referendum campaign.