Sir David Murray, the Rangers FC chairman, made a dramatic intervention in the run-up to the Glenrothes by-election claiming that the Union was essential for the survival of Scottish business.
Murray said an independent Scotland would lose out on UK defence deals such as the 3bn contract for the UK's biggest aircraft carriers being built at the Rosyth Dockyard.
Speaking to Scotland on Sunday, Murray, a major employer in the constituency, said: "In an independent Scotland these jobs would go."
Murray, whose business empire is worth an estimated 720m and employs 5,000 people, said the Scottish economy depended on enterprises such as his own Murray Group, the property specialists Miller Group, the engineering company Jim McColl Associates, the publishers DC Thomson and Stewart Milne, the Aberdeen-based developer.
"Who really drives the economy?" Murray said. "It is the private sector firms like Jim McColl, Miller, Thomson, Stewart Milne and Murray. Taking these groups together, they are starting to employ as many people as these banks.
"These are privately owned companies, Scottish funded and family owned. They are surviving and getting business and the reason is that we are an integral part of the Union.
"I am not political, but I strongly believe in the continuance of the Union. If we don't back these companies then we might as well go home."
Murray was speaking after meeting with Gordon Brown after the Prime Minister visited one of the entrepreneur's business interests while campaigning in Fife.
"I'm not political. I'm for the Murray party," Murray said before adding that his reason for speaking out as the by-election approaches was his passionate belief in the Union.
While he did not explicitly back Labour, Murray's remarks will come as a blow to Alex Salmond, whose arguments for independence have been dented by the global economic crisis.
In the aftermath of the RBS and HBOS bailouts, Murray pointed out that the Scottish banking system relied on support from the UK Government.
He also said he believed that the support of the UK Government was essential as Scotland's tax-take falls as the recession bites.
The Rangers chairman suggested that an independent Scotland would struggle to land major Ministry of Defence contracts such as the aircraft carrier order, which will support more than 3,000 jobs at BAE's Govan and Scotstoun yards in Glasgow, and sustain 1,600 jobs at Babcock Marine's naval dockyard at Rosyth on the Firth of Forth.
"There has to be a question mark over the placement of the military work and the jobs there," Murray said.
His remarks were endorsed by the Prime Minister, who was visiting the high-tech Glenrothes-based Brand-Rex company that has recently won a 5m contract to fit the aircraft carriers with IT cabling.
The Murray Group has bought a stake in the company, which also supplies the American Navy. Brown said: "This is all about the Union and the benefits of being in the Union. This is a case when the British Navy is able to order two new aircraft carriers and we have always known that the SNP's defence policy is in complete ruins."
Brown went on to renew his attacks against the SNP's so-called 'Arc of Prosperity' argument, which claimed that an independent Scottish economy would flourish in a similar manner to the Icelandic, Irish and Norwegian economies.
With the Irish economy in recession and Iceland in meltdown, Brown said: "We are stronger as part of the UK. The banks are now recapitalised and 80% of sales of HBOS and RBS were in England and less than 20% of the people work in Scotland themselves, and their future depends on being part of the UK.
"We share the risks, resources and rewards. That's what being part of Britain means. Through the big economic downturn we are stronger together than we would be apart."
A spokesman for Salmond said:
"Scottish companies, including Sir David's, would be free in an independent Scotland to compete for any contracts on the basis of price and quality. Many Scots engineering firms have already proved themselves to be outstandingly successful globally on that basis."