Bill Munro, founder and non-executive director of Barrhead Travel, distributed an email to 697 of its 800 staff and directors warning of "impending disaster should there be a Yes vote" in the referendum on September 18.
"I have been asked by many staff what my views of the referendum are," Mr Munro said.
"Below and attached are a copy of the facts, not the lies being put out by the SNP."
A report by banking expert Robert Lyddon on the fiscal implications of Scottish independence was attached.
Mr Munro continued: "For Barrhead Travel and most if not all travel companies it will be a complete disaster, especially with branches in England and Ireland.
"37% of our business comes from England and we would not be able to trade outwith Scotland due to Scotland being outwith the EU and English regulations for at least three years.
"The devil is in the detail and as the weeks pass and more is discovered the magnitude of the impending disaster should there be a Yes vote becomes ever more apparent."
The letter was leaked on social networking site Twitter, prompting Mr Munro to release a statement distancing his views from the company and its employees, who he said have the right to express their own views on the referendum.
In the statement, he said: "In response to an increasing number of questions from Barrhead Travel employees regarding my views on the referendum, I decided to share my personal opinion which is that independence would not be good for Scotland, its businesses or its consumers.
"One of my main concerns is that of the financial protection of the Scottish travelling public which would be under threat in an independent Scotland. Second to that is the uncertainty of cross border trading which I believe will be impacted negatively, a concern especially for businesses with interests outside of Scotland.
"The opinions expressed are my own and not those of the company. Just as I have the right to express my own opinion, I respect that is true of everyone in Scotland, including the 800 employees of Barrhead Travel who will make their own views clear throughout the democratic process."
Speaking on behalf of Better Together, East Renfrewshire Labour MP Jim Murphy said: "Barrhead Travel is yet another example of an employer in Scotland saying that being part of the UK is better for their business. Having access to a single UK market of 63 million people, rather than five million, means Scotland's firms have more opportunities to thrive and employ more people. Where is the sense in putting a barrier between Scottish businesses and their customers elsewhere in the UK?
"Businesses still aren't being told what would replace the pound and how we would get back into the EU or on what terms. Asking Scotland's employers to hope that it will be alright on the night isn't good enough.
"Being part of the UK means we can have the best of both worlds - a strong Scottish Parliament, with the guarantee of more powers, backed up by the strength, security and stability of being part of the larger UK. Independence puts that at risk.
"Barrhead Travel is a real Scottish success story to be proud of. The nationalists' response to the company saying they want to stay part of the UK is a new low. This is the worst type of negative politics from Yes campaigners."
Gordon MacIntyre-Kemp, chief executive of pro-independence group Business for Scotland, said: "As recently as last week Bill Munro, of Barrhead Travel, was on STV saying he wanted, as many people do, to know what's best for Scotland, and like other voters that's what he was looking for.
"He went on to say that he didn't think there were currently sufficient answers to the many questions he still has.
"Business for Scotland believes the business case is clear. The idea that in the event of a Yes vote, for example, there would be restrictions on trade, as outlined in the memo, doesn't make any sense. Day in, day out there is free trading taking place between independent countries.
"Business for Scotland would be happy to outline the overwhelming case for independence to Bill Munro and let him see why we believe it is the business opportunity of a lifetime."