Mr Ashley, owner of Newcastle United and also a shareholder at Rangers, has steadily been boosting the fashion offering at the group in recent years and already has 160 shops under the USC, Cruise, Republic, Van Mildert and Flannels fascias.
Previously, Mr Ashley has considered investing in House of Fraser, where his business associate Sir Tom Hunter has a stake, but that deal appears to be dead as the department store owner eyes an initial public offering this year and remains in talks with French company Galeries Lafayette.
Shares in Debenhams have plunged in recent weeks on the back of a profit warning on December 31 that came after poorer than anticipated festive trading. Chief financial officer Simon Herrick then stepped down a few days later.
Sports Direct said it had built up the stake on January 8 and 9 when shares in Debenhams were changing hands for between 75.5p and 76.6p.
To acquire the 56.8 million shares, equivalent to a 4.63% stake, Mr Ashley's business is likely to have paid out a sum of between £43 million and £46m.
Yesterday Sports Direct confirmed it was interested in collaborating with Debenhams at an operational level.
It said: "This acquisition of shares has taken place without the prior knowledge of the Debenhams board of directors, but Sports Direct has communicated to Debenhams' board its desire to work together and its intention to be a supportive shareholder."
Debenhams responded by saying it is "open minded" about operational opportunities to improve its performance alongside its own initiatives. A meeting between the executive teams of both businesses is thought likely to take place in the coming weeks.
Cantor Fitzgerald retail analyst Freddie George said the developments did not mean Debenhams was "in play" from a takeover perspective.
He pointed out Sports Direct had taken stakes in the likes of Blacks Leisure, JJB Sports and JD Sports in the past.
Mr George said: "In our view one should not read too much into this new investment."
There was speculation Sports Direct wants to establish concessions for some of its fashion brands, such as Firetrap, Full Circle, Kangol and Soviet, within the 156 Debenhams UK outlets.
However Sanjay Vidyarthi, from Liberum Capital, said that scenario did not seem likely and said: "A full takeover or attempt to merge the businesses in any way seems unlikely to us and the wording of the statement suggests a more collaborative approach.
"Between them, the two retailers have a lot of space and a lot of brands. There would be some overlap between the Sports Direct and Debenhams customer demographic, but less so with premium fashion, in our view.
"We would not expect those brands to be sold through Debenhams and it is unlikely that many of Debenhams' own label, Designers at Debenhams or third party brands would be suitable for either Sports Direct or its premium fashion stores."
That view was echoed by retail analyst Nick Bubb who said: "The way forward for Debenhams ought to be to move upmarket a bit, discount less.
"Dragging Debenhams downmarket and turning it into even more of a discount store may not appeal to retail purists."
Shares in Debenhams rose more than 7% in early trading but fell back to close the day up 4.1p, or 5%, at 85.65p.
Sports Direct was down 8.5p, or 1%, at 747.5p.