"Contrary to rumours in Israel, there is no intention to transfer to the Vatican sovereignty or ownership on the Tomb of David or the Cenacle," Israel's ambassador to the Vatican, Zion Evrony, said in a speech obtained on Wednesday.
The Cenacle is the reputed scene of Jesus's last supper, and the pope is planning to hold a mass on the site on Mount Zion near the walls of the Old City.
Jews revere the site as the tomb of Kind David, which is on the ground floor of the same building. It is also important for Muslims.
The site is currently part of long-running negotiations over Holy Land property between Israel and the Vatican, which hopes to be given the right to use the site.
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Hundreds of ultra-Orthodox Jews on Monday held a protest near the site, demanding that Israel retain sovereignty. They are also planning another demonstration on May 22 -- just three days before Pope Francis arrives.
Francis is travelling to Israel, Jordan and the Palestinian Territories between May 24 and 26.
Israeli President Shimon Peres told an Italian newspaper during a visit to the Vatican last year that a compromise had been reached on the Cenacle, and that "99 percent" of the issues concerning the site had been addressed.