Bookmaker William Hill said the bet – the largest political gamble it has taken – would bring the unnamed man a total profit of £36,666 if there was a No vote in September 2014.
“We have never taken a bigger bet than this on any political subject,” said spokesman Graham Sharpe.
“The man, in his late 50s, with a pronounced Scottish accent, and a stranger to staff, struck one bet of £30,000 at odds of 1/5, then returned to place another, of £70,000 at the same odds, before placing a third, of £100,000 at odds by now shortened to 1/6.”
The bet was placed in Glasgow.
An SNP spokesman said the gambler had only wasted his money, adding: “This underlines that a No vote is the real gamble with Scotland’s future – because it means more Tory governments the people of Scotland didn’t vote for.”
Polls suggest most people in Scotland will not back independence, but there are still 450 days to go before the vote.
In May, William Hill took a bet of £5,000 on a Yes vote at odds of 3/1.
The current odds are 1/6 for a vote against and 7/2 for independence.