The 250-strong group from Stornoway High on Lewis, including the entire Kirk Session, left claiming it was departing from Biblical teaching on various issues including allowing gay clergy.
The breakaway group had become members as both the Kirk and Free Church hold their annual assemblies in Edinburgh.
The Stornoway faction is thought to be the biggest group to have quit over the Kirk stance on gay ordination. More than 200 worshippers remain at the church under an interim moderator.
The announcement will be seen as a clarion call for hardline traditionalists in the Church of Scotland who are struggling with the Kirk's plan to continue to allow congregations and Kirk sessions to choose whether to have a gay minister in a civil partnership.
Eight former Church of Scotland ministers have joined the Free Church in recent years, as well as many elders and members. A Church of Scotland source said it is expected only a small number of ministers will leave over the Kirk's move towards greater acceptance of gay clergy and congregations are thriving under new leadership where ministers or church members have left.
The issue that has divided Kirk worshippers since the Rev Scott Rennie, who is gay, was appointed to an Aberdeen church in 2009 could lead to more ministers and worshippers quitting. Since then 13 ministers have left the Kirk.
Christopher Martin, former Stornoway High session clerk, said: "We have always said we wanted to continue in a presbyterian setting, and having looked at a number of possibilities it became clear the most favourable and accommodating was to join the Free Church. Sadly, our congregation could simply not identify with the general direction of the Church of Scotland is headed in, and the sensible option was to leave."
The Rev Iver Martin, minister of Stornoway Free Church, said: "No-one is pleased with the current situation in the Scottish churches, but I am glad that the High fellowship has joined our denomination.
Free Church minister in Inverness and former moderator the Rev David Meredith said he has been chairing a working group set up to deal with requests from congregations seeking to join the Free Church.
The new Free Church congregation - who are without a minister - are worshipping in a primary school in Stornoway. The High was previously the Kirk's biggest congregation in the Western Isles.
The Free Church said it has increased its membership over the past five years including a "notable increase in people under 30 attending services".
A spokesman for the Presbytery of Lewis said the church was saddened by the move.
He added: "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."
Meanwhile, the Kirk is to examine plans that would bring to an end the tradition of the annual General Assembly. Instead, it would be held every two years.
The one-year term of office of the Moderator would last for two years.