"As far as we can ascertain this is the biggest political bet ever struck," said Graham Sharpe, media projects director at William Hill.
The bet is twice the 200,000 pounds, also put on a "no" vote, staked by a Scottish man earlier in the independence campaign. By way of comparison, a customer similarly gambled 200,000 pounds on David Cameron, the current prime minister, winning the leadership of his Conservative Party in 2005.
Read MoreScottish independence: Is UK government 'blethering'?
Scots will vote in a referendum on Sept. 18 on whether to leave the United Kingdom. Polls currently suggest roughly 40 percent of Scots will vote against independence and 30 percent will vote for, but many voters remain undecided.
William Hill believe they will turn over more than one million pounds in bets on the independence outcome.
The bookmakers said they had shortened their odds on a "no" vote to 1/5, suggesting this was now more likely to happen than previously thought. Their odds on a "yes" vote stand at 10/3, meaning winners getting 10 pounds back for every three they bet.
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