It was not immediately clear how long the closure of the church, announced by Christian officials at a news conference and which began at around noon, would last.
The church is considered the holiest site in Christianity, built where Christians believe Jesus was crucified, buried and resurrected, and is a major pilgrimage site.
"As a measure of protest, we decided to take this unprecedented step of closure of the Church of the Holy Sepulchre," Greek Orthodox, Armenian Apostolic and Catholic leaders said in a statement.
They said recent Israeli measures seemed to be "an attempt to weaken the Christian presence in Jerusalem."
Christian leaders have been angered over attempts by Israeli authorities in Jerusalem to enforce tax collection on church property they consider commercial, saying exemptions only apply to places of worship or religious teaching.
Separately, Christian leaders say legislation being considered by Israel's government would allow church property to be expropriated.
"This abhorrent bill is set to advance today in a meeting of a ministerial committee which if approved would make the expropriation of the lands of churches possible," the statement said.
"This reminds us all of laws of a similar nature which were enacted against the Jews during a dark period in Europe." Confused tourists stood in front of the church's closed doors in Jerusalem's Old City after the announcement as tour guides sought to explain why they could not visit.
One man knelt and prayed as workers began putting metal barriers around the entrance.
Jerusalem Mayor Nir Barkat said in a statement that the city was due 650 million shekels ($186 million, 152 million euros) in uncollected taxes on church properties, which he called "illegal and irrational."
Barkat stressed that the Church of the Holy Sepulchre and all other churches were exempt from the taxes and would remain so, with the changes only affecting establishments like "hotels, halls and businesses" owned by the churches.
Christian leaders say the measure jeopardizes their ability to conduct their work, which includes not only religious but also social services to those in need.
A separate bill seeks to allay fears of Israelis who live in homes on lands previously held by the Greek Orthodox Church and which were sold to private developers, according to Rachel Azaria of the centrist Kulanu party, who is proposing the legislation.
Recent land sales by the Greek Orthodox Church have drawn fire from both Israelis and Palestinians.
According to Israeli media, the foreign ministry has been critical of the Jerusalem mayor's decision on church taxation, with officials saying the move was harmful to a decades-long status quo.
A Russian tourist in her 20s who only gave her name as Elona said "it is very disappointing" that she could not visit the church on Feb. 25.
"It is one of the main religious attractions, and to us it was very important to visit it because it is our first time [here]," she said.
She said she does not feel "her mission is accomplished" as she was only visiting for a few days.