Mr Ashley's share sale comes less than a year after he raised £100 million by offloading a 4% holding in Sports Direct, sparking speculation over his plans for the cash haul.
The move was announced a day after recent FTSE 100 Index entrant Sports Direct reported yet more robust trading figures, with a 15.1% sales surge in the nine weeks to September 29.
He is not the only retail boss to cash in after managers at online fashion firm ASOS sold 1.88 million shares worth £94.1 million - 2.3% of the company - in the wake of a 37% jump in annual profits.
ASOS chief executive Nick Robertson, finance director Nick Beighton and other executives sold shares from a 2009 share incentive scheme, while Mr Robertson also separately raised £42 million from selling shares from another bonus plan.
But Sports Direct saw its stock price fall as much as 9% at one stage today after Mr Ashley's sale of 16 million shares for 662.5p, before recovering a little to stand 5% lower.
Rumours had swirled around his share sale earlier this year, with fears that Mr Ashley was calling the top of the share price.
But the stock has continued to surge since February, up more than 70% so far in 2013 and earning the group promotion into the FTSE 100 Index last month.
Independent retail analyst Nick Bubb said: "T here was a lot of fuss about him cashing in, but these moves only help to improve liquidity over time and the sale yesterday... seems to have been taken more calmly."
Mr Ashley caused ill-feeling in the stock market after Sports Direct shares plunged following its flotation in February 2007, which saw him sell a 43% stake for more than £900 million.
Having listed at 300p, the shares went as low as 33p after a string of profit warnings.
But a reliably strong performance since then has helped the group gain the City's confidence as Sports Direct benefits from the demise of rival JJB Sports and a recovering retail sector, as well as ongoing investment and the positive impact on employees of a lucrative bonus scheme.