The charity, which fundraises for conservation and restoration work at York Minster, said in a statement to Premier on Thursday that the Duke of York has resigned as a trustee following a decision to step back from public life.
"However following his decision to step back from public life, and recognising that the Fund needs an active patron to champion its work, HRH Prince Andrew has decided to resign from this role.
"The trustees of the Fund would like to thank the Duke of York for all his work as Patron over the years."
This comes after revelations of Prince Andrew's alleged involvement with Jeffrey Epstein, a financier who died in prison in August as he waited for his trail on sex trafficking charges.
Virginia Roberts Giuffre claims in 2001, at the age of 17, she was trafficked by Epstein and pressured into have sex with Prince Andrew.
In a BBC's Newsnight programme, the Duke of York denied that he has ever had any form of sexual contact or relationship with Giuffre.
The York Minster Fund resignation isn't the first charity the prince has left since the allegations were made public.
Last month, he resigned from his roles at the Outward Bound Trust and The University of Huddersfield.
Stay up to date with the latest news stories from a Christian perspective. Sign up to our daily newsletter and receive more stories like this straight to your inbox every morning.