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St Matthew's Church in Stafford St this week voted to disaffiliate from the church.
Bishop of Dunedin the Rt. Rev Steven Benford confirmed the move after inquiries from the Otago Daily Times.
He said at a special meeting on Monday the St Matthews congregation in Dunedin voted to disaffiliate, "a result of the General Synod resolution on the blessing of same-sex civil marriages passed in May 2018''.
Dr Benford said: ''It is with a heavy heart that we have heard the decision of a portion of St Matthew's parish to disaffiliate from the Anglican Church.
"We have been continuing to talk and pray with the leadership at St Mathew's over this decision.
"Although we are saddened that part of the congregation is no longer going to be within the Diocese of Dunedin, we are committed to the future of the parish both pastorally and for its future mission in Dunedin.
Earlier this year St Matthew's vicar Stu Crosson wrote the move to allow the blessing of same-sex marriages "appears to me to be a step into false teaching, contrary to the unified witness of scripture, a denial of what it means to be faithful, human, image bearers, in our maleness and femaleness and as such an idolatrous step away from our God.''
He said to bless something God called an abomination and the apostle Paul described as "inviting the wrath of God'' seemed destined to invite the judgement of God upon the church.