Sir Andrew Cook, who has donated more than £1.2m to the party,told The Timesthe country could "sleepwalk to disaster" if it made such a move.
The engineering firm chairman said at least one of his factories was almost "entirely dependent" on access to it.
Sir Andrew backed the Remain campaign in the EU referendum.
He told the newspaper that the "economic arguments of staying in the single market are overwhelming" and it would be a "catastrophe" if the country left.
"It is very difficult to make a political donation to a party when, although I support it ideologically, I do not believe that my interests and my ideology are ad idem with the principal Brexiteers," he said.
Theresa May has insisted that she wants firms to have the "maximum freedom to trade with and operate in the single market".
But the prime minister is due to confirm in a speech later this month that the UK will have two fundamental red lines in its Brexit negotiations - control of its borders and freedom from the European Court of Justice.
She has pledged to trigger Article 50 of the Lisbon Treaty - so getting leaving talks with the EU under way - by the end of March. These can take up to two years, unless an agreement is reached to prolong the process.
In November last year, the Conservatives reportedmore than £2.8m in donationsin the three months after the EU referendum - the highest amount of the major parties.
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