UK insurance and pensions heavyweight Standard Life became the first major company to warn it could move parts of its business out of Scotland if Scots split from the United Kingdom.
The comments are a blow to Scotland's independence movement.
The company, which has been based in Scotland for 189 years, said today that it had a contingency plan to partly move from Scotland, potentially putting 5,000 jobs at risk.
Chief executive David Nish said it had started setting up registered companies in England "as a precautionary measure" into which it could transfer operations if Scots voted on September 18 to end a 307-year union with England.
Nish said the steps were necessary due to uncertainty over how an independent Scotland would work, including what currency it would use and if it would achieve its goal of European Union membership.
A war over currency has ignited the independence debate in recent weeks, with Scottish leader Alex Salmond wanting to share the pound in a currency union but the rest of the British parties rejecting this proposal.
Nish stressed Standard Life was maintaining a position of "strict political neutrality".
"We have started work to establish additional registered companies to operate outside Scotland into which we could transfer parts of our operations if it was necessary to do so," Nish said on a conference call today.
"This is a precautionary measure to ensure continuity of our business' competitive position and to protect the interests of our stakeholders," he added.
Scotland is home to the second largest financial services industry in the United Kingdom, employing about 85,000 people directly and another 70,000 in support companies.
Standard Life, with 5,000 staff in Scotland, currently manages more than £244 billion of assets for its clients, around 90% of whom are based outside Scotland.
So far few businesses have publicly stated their plans for what they would do if Scotland did become independent as the likelihood of a "Yes" vote had seemed remote.
But with the nationalists starting to gain ground in the polls, with about 37% support compared to 47% opposition to independence, increasing numbers of business leaders have started to point out possible risks.
A poll by executive search firm Korn Ferry released this week found 65% of chairmen of 32 FTSE 100 companies said it would be bad for business if Scotland became independent.
Meanwhile, Scotland would face a significant but not unsurpassable challenge if voters back independence in a referendum later this year, ratings agency Standard & Poor's said today.
While an independent Scotland would have the attributes of a wealthy investment-grade economy, it would face high levels of public debt, sensitivity to oil prices and potentially limited monetary policy flexibility, S&P said.
"At the same time, Scotland's external position in terms of liquidity and investment could be subject to volatility should banks leave," the ratings agency said in a statement.
"On the other hand, if this were to happen, it could bring benefits in terms of reducing the size of the Scottish economy's external balance sheet, normalising the size of its financial sector, and reducing contingent liabilities for the state," it added.