A stock exchange announcement on Tuesday showed that Sports Direct has raised its interest in Debenhams from 19% to 21%, the latest in a series of increases.
Mr Ashley's intentions in boosting his firm's holding have been the subject of speculation, although few have tipped the billionaire to attempt a full-blown takeover.
Sports Direct has in the past said it simply wants to be a "supportive stakeholder" in Debenhams, and to create value in the interests of shareholders of both firms.
The retailer began building its stake in Debenhams in 2014 and last made a raid on its shares in June.
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Sports Direct has built its position through a combination of share options and contracts for differences.
Newcastle United owner Mr Ashley has a history of building up positions in rival retailers, having previously bought stakes in JD Sports and privately-owned House of Fraser.
Sports Direct also snapped up a 27% stake in French Connection earlier this year, raising fresh questions over the chain's future.