The head of US-based investor Pimco, Mohamed El-Erian, said it would raise the UK’s cost of borrowing in markets.
However, a group of 55 British business leaders have written an open letter to the Times throwing their weight behind Mr Cameron’s strategy.
Mr Cameron is due to speak at the World Economic Forum in Davos later.
“We need a new relationship with the EU, backed by democratic mandate,” said the group of top bosses, who include the chief executives of B&Q-owner Kingfisher, mining group Xstrata, electricals retailer Dixons, the London Stock Exchange and beverages maker Diageo, as well as the chairman of engineering firm Rolls Royce.
The executives complained about the red tape burden imposed by Brussels, and claimed it was the right moment “to push for a more competitive, flexible and prosperous European Union that would bring more jobs and growth for all member states”.
The UK’s biggest business organisation, the CBI, also expressed support for the mooted in-or-out referendum.
Speaking on the BBC’s Hardtalk programme, Mr EL-Erian – who heads the world’s biggest investor in bonds, based in California – said the UK would “certainly suffer the consequences” if it exited the EU, including lower growth and lower investment.
But he said the uncertainty generated by the possibility of an EU exit years in the future would also be damaging.
“People like us start putting in an uncertainty premium,” said the US-based fund manager.
“If we’re going to make investment decisions, the uncertainty premium associated with that goes up when you’re not sure what the relationship between Britain and Europe will be.”
If it goes ahead, the referendum is due to be held between 2015 and 2017.
David Sproul, the UK boss of Deloitte, said: “The Europe debate does not help to create certainty.
“When I talk to US clients who have not been immersed in the European debate as we have, they say that what they need is clarity. There is no question it will impact business – it will hit investment into the UK.”