The restaurant, which one cardinal labelled "perverse" when it opened just metres away from St Peter's Basilica in January, will provide up to 1,000 homeless people free meals every Monday.
The branch - nicknamed locally as McVatican - has agreed to work with Medicina Solidale, a local charity that provides medical care to the homeless, to help distribute food to those living on the streets nearby.
The lunch will consist of a double cheeseburger, apple pie and bottle of water.
"Providing a meal guarantees an appropriate supply of proteins and vitamins to so many men and women who live on the street," said Lucia Ercoli, director of the charity. "I am very happy with this agreement with McDonald's."
The initiative comes after cardinals and residents complained that the fast food giant was out of place in the historic city.
Last October, Cardinal Elio Sgreccia said that the property would be "better to use those spaces to help the needy of the area, spaces for hospitality, shelter and help for those who suffer, as the Holy Father teaches."
Local media has reported that McDonald's is paying $32,000 a month to rent the premises.