Keith Cochrane, the chief executive of engineering giant Weir Group, said "the costs of independence are guaranteed but the benefits are uncertain" and warned his company would consider moving its HQ from Glasgow if negotiations to set up an independent Scotland, following a Yes vote in September, appeared to be going badly.
He spoke out after Weir Group, which was founded in 1871 and employs 15,000 people in 70 countries, published new research into the impact of independence.
The study, by leading independent analysts Oxford Economics, concluded firms were likely to face higher taxes, higher borrowing costs and new barriers to trade if Scotland left the UK.
At Holyrood, Deputy First Minister Nicola Sturgeon dismissed the concerns but said she was due to meet senior executives and hoped to reassure them.
Meanwhile the Yes campaign also welcomed a leaked internal memo saying broadcaster BSkyB, which employs 6400 people in Scotland, had no plans to leave the country in the event of a Yes vote.
Unveiling the new study - a response to the Scottish Government's White Paper on independence - at Weir Group's Glasgow base, Mr Cochrane said: "As a political document, the Scottish Government's White Paper paints a picture of independence as being a risk-free option with only potential benefits.
"However, voters should be aware that what they are being asked to say Yes to carries substantial risks to our economy and therefore to the quality of life of millions of people."
He said the economy was a "vitally important" consideration ahead of September's vote, adding: "I share the strong emotional tie we all have to our country, but in the referendum debate heads must rule hearts."
The Oxford Economics study said there was "little clarity" on an independent Scotland's possible currency. But it warned that under any of five potential currency arrangements - including the Scottish Government's proposal to share the pound and creating a new currency - an independent Scotland would face substantially higher borrowing costs, which would affect businesses.
Mr Cochrane said: "The independent analysis we commissioned highlights one thing for sure - there will be additional costs.
"There is no such thing as risk-free independence."
He said the company would not consider moving its headquarters from Scotland before the start of formal independence talks, but added: "Then we will be able to see the full picture. Clearly at that point we will need to review the situation."
A number of other leading Scots firms, including insurer Standard Life, have already voiced concerns about independence.
Mr Cochrane's comments were welcomed by Alistair Darling, the head of the pro-UK Better Together campaign.
"Yet again it is clear from a company employing hundreds of Scots that being part of the UK is good for jobs here and it keeps down costs for families in Scotland," he said.
Finance Secretary John Swinney and Deputy First Minister Nicola Sturgeon - who was challenged over Mr Cochrane's comments when she stood in for Alex Salmond during First Minister's Questions - both dismissed his concerns.
Mr Swinney said "International comparisons show that independent countries of Scotland's size have considerably larger manufacturing sectors than the UK and are able to offer more support to their manufacturing companies than Scotland is currently able to do."
He welcomed comments by BSkyB's director of corporate affairs, Graham McWilliam, who said in a memo to staff: "We like being in Scotland. And we have no plans to change that."
The memo - confirmed by the company - said BSkyB would not take a position on independence but "let the people have their say".