The six ministers campaigning for the UK to leave the EU, including justice secretary Michael Gove and leader of the Commons Chris Grayling, will be unable to use official briefings to prepare campaign speeches.
Speaking to the BBC, Northern Ireland secretary Theresa Villiers, another minister campaigning for Brexit, said: "The Leave campaign is the underdog, no doubt about that. Much of the establishment is lined up in favour of EU membership, as they have been in the past."
Her statements come after cabinet secretary Sir Jeremy Heywood, the UK's most senior civil servant, said the EU referendum guidance makes clear the government isn't neutral on the question of the UK's EU membership.
"It will not be appropriate or permissible for the Civil Service to support Ministers who oppose the Government’s official position by providing briefing or speech material on this matter," the guidance said. "This includes access to official departmental papers, excepting papers that Ministers have previously seen on issues relating to the referendum question prior to the suspension of collective agreement. These rules will apply also to their special advisers."
The guidance said civil servants "can communicate government policy including in making the case for Britain to remain in a reformed EU".
It comes after Prime Minister David Cameron spoke in parliament yesterday, saying that the government's official position was that the UK should remain in a reformed EU.
The briefing paper added that civil servants and special advisers should not give ministers campaigning for Brexit any briefing or speech material on the matter, except papers that the ministers have already seen.
Although the government has taken a firm position, the Conservative party is officially neutral in the referendum debate.