The 77-year-old stepped down with immediate effect to fight “false allegations about his private life”.
His tenure included West Ham’s unpopular relocation from Upton Park to the London Stadium in 2016.
In a statement, he said: “After very careful consideration and with a heavy heart, I have decided to resign as joint-chair and director of West Ham United FC with immediate effect.
“This has been an incredibly painful decision to make, but it is one made out of love, respect, and responsibility toward a football club and a fan base that deserve absolute unity and focus moving forward.”
Interim chief executive officer Karim Virani, reporting into the board of directors, will continue to be responsible for leading the club’s day-to-day operations.
A club statement said: “West Ham United can confirm that Joint-Chair David Sullivan has stepped down from his position with immediate effect.
“Mr Sullivan has also resigned as a director of both WH Holding Limited and West Ham United Football Club, having been made aware of the impending publication of serious historic allegations.
“It is understood none of the allegations relate to West Ham United or any of its operations.”
Sullivan, along with his business partner David Gold, took control of West Ham after selling Birmingham.
Baroness Karren Brady also joined the club as vice-chairman.
Gold died in 2023 aged 86, while Baroness Brady left her role before the end of a challenging season which ended in relegation from the Premier League.
“None of these allegations relate to my more than 30 years in football,” added Sullivan.
“West Ham United has been one of the greatest passions and privileges of my life. I care deeply about the club, its supporters, its players, its staff, and its future.
“At what is already a challenging and important time for the club, I refuse to allow personal matters concerning me to become an unnecessary distraction or a source of instability.”
West Ham say they will give “an update on the future structure of the board of directors in due course, but will make no further comment at this time”.
Czech businessman Daniel Kretinsky bought a 27 per cent stake in the club in 2021 and is reportedly set to increase his stake.