A cross-party group of MPs have said they are confident that a new campaign would pressure Gordon Brown into holding a referendum on the EU Treaty.
A 15ft (4.5m) inflatable ballot box was used to launch the campaign
Ex-Labour ministers Kate Hoey and Frank Field are among MPs who claim voters were promised a say by the government.
They will use a website, cinema adverts and events to put their message across.
A referendum on the EU constitution was pledged in 2005, but Mr Brown says the treaty is different to the abandoned constitution so no poll is needed.
Supporters of the I Want a Referendum campaign claimed they had the support of about 50 Labour MPs and the sympathy of a further 60 to 70.
Labour MPs at the project's launch suggested that if 100 of their colleagues signed a Commons motion calling for a referendum, it would be "game, set and match", and the prime minister would be forced to organise a vote.
Government whips are so alarmed by the potential rebellion that they are contacting MPs to assess the size of any potential revolt, Labour sources have claimed.
Mr Brown has said that Britain has got its way in the treaty's proposals for key areas such as justice and security - and as long as that does not change there should be no need for a poll.
"If we succeed in achieving our red lines in all the detailed negotiations, there will, in my view and in the government's view, be no need for a referendum," Mr Brown told the BBC on Monday.
The "red lines" he refers to include Britain keeping control of its foreign policy, tax, benefits and criminal justice, and being able to opt out of a charter of fundamental rights.
But the prime minister's stance has not convinced Derek Scott, vice-chairman of Eurosceptic pressure group Open Europe, who is chairing the referendum campaign.
"It's going to be, quite clearly, a difficult campaign," he told the BBC News website, acknowledging that some voters found it hard to engage with complicated EU policy documents.
"We're trying to persuade people - but not by going into the details of Article 4 or Article 5, because that isn't something which is going to resonate with them very much.
"But I do think they understand the basic issue here, which is a matter of trust, and there is a great deal of disillusionment with politicians of all parties."
Mr Brown argues no referendum is needed to approve the treaty
Meanwhile Mr Field, a former social security minister, added that most MPs "are returned with votes of only between 50% and 60% of the electorate".
"There's a huge gap of cynicism between the electorate and us.
"They don't think we're interested in their views, and that we've got our own little agenda, and here is a chance of narrowing that divide. We should actually seize it with both hands."
Organisers of the campaign, launched next to the Houses of Parliament, used an inflatable ballot box measuring about 15ft (4.5m) to get their message across.
The initiative is also backed by Labour's Gisela Stuart and Graham Stringer, Conservative MPs Michael Gove, Nick Herbert, David Heathcoat-Amory and Greg Hands, as well as Mike Hancock of the Liberal Democrats.