The Kirk Session of elders at the High Church in Stornoway, Lewis, has walked out and an estimated 200 members are expected to follow.
Breakaway worshippers are forming a new congregation called the Stornoway High Fellowship.
It will gather this weekend and will hold services in a hired hall until further notice.
Christopher Martin, the former session clerk at Stornoway High and the spokesman for the new congregation, said: "Everyone is very sad at having to leave.
"This has been going on for so long and people feel there is no option but to leave friends and the building in which they have worshipped for many years."
The debate, which has been ongoing since the openly gay Reverend Scott Rennie was appointed to an Aberdeen congregation in 2009, will be raised at the General Assembly next month.
A spokesman for the Presbytery of Lewis said: "We are saddened that a large number of members and adherents of Stornoway High Church have indicated their intention to leave the Church of Scotland.
"However Lewis Presbytery is still of the opinion that there is an essential place for the High Church congregation in ministry and mission within Lewis, a ministry which stretches back more than 100 years.
"Our priority as a Presbytery is to take the necessary steps to fulfil that objective through those who have chosen to remain."