MASH Holdings, a vehicle wholly owned by the Newcastle United owner and Rangers investor, disposed of 25 million shares at 400p each.
The sale was handled by Goldman Sachs and reduced the MASH Holdings stake in Sports Direct to 64.4%.
It is the first time Mr Ashley, who does not take a salary, has sold shares in the retail business since its stock market flotation at 300p a share in February 2007.
Then he was estimated to have made around £929m in one day. There was no indication given as to what the entrepreneur plans to do with the latest £100m, although he has been linked with a bid for House of Fraser in the past.
Analysts speculated the move may have been motivated by financial planning ahead of the tax year-end, or pressure to improve the stock's liquidity. Shares in Sports Direct closed down 21.5p, or 5%, at 409p.
Sports Direct said: "In connection with this disposal, MASH Holdings Limited has agreed not to sell, or otherwise dispose of, any interest in any ordinary shares of the company, or enter into any transaction having a similar economic effect, for a period of 365 days from February 25, 2013."
Sports Direct, which has 400 UK stores and includes Cruise and USC in its portfolio, has been one of the success stories in a difficult high street environment.
Last week it posted a 21.2% increase in third-quarter sales to £589.5m.