Duffen has decided to step down with immediate effect, following a turbulent time at the club.
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A statement on City's official website read: "The club has received a letter of resignation from Paul Duffen which has been accepted and is effective immediately.
"Paul's departure is on a totally amicable basis and he leaves with our best wishes as a friend of the club and its directors.
"The club wishes to express its gratitude to Paul for his outstanding contribution as chairman during a period of unprecedented success for Hull City AFC."
Wednesday had seen the club deny that manager Phil Brown had been sacked, following fevered press speculation.
City had also been warned that they could face serious financial problems if they are relegated from the Premier League this season.
The club's accounts, filed five months late to Companies House, say that in the event of relegation the club will need to generate a £23million surplus just to meet their existing liabilities.