A club statement said: "The club has received a letter of resignation from Paul Duffen, executive chairman of Hull City AFC and Superstadium Management Company Ltd, which has been accepted and is effective immediately."
There had been speculation over the future of both Duffen and manager Phil Brown in recent days, with the club taking the unusual step on Wednesday of denying reports that Brown had been sacked.
Hull issued a statement after several leading bookmakers stopped taking bets on Brown being the next Premier League manager to lose his job.
Hull are currently in the Premier League relegation zone, a point shy of safety, with only West Ham and bottom club Portsmouth below them in the table.
The Tigers' next match sees them away to Burnley on Saturday.