Up to 34 people will be offered a bed every night at the facility which is just steps from St Peter's Square.
Pope Francis's chief charity guru, Monsignor Konrad Krajewski, inaugurated the shelter.
The building is owned by the Jesuits and the shelter is operated by Mother Teresa's Sisters of Charity religious order.
A statement from Mgr Krajewski's office said all expenses are paid for by donations and the proceeds of papal parchments sold by his office.
It is the latest papal initiative to help the homeless around the Vatican. A few months ago Mgr Krajewski's office opened showers and a free barber shop for the needy near St Peter's Square.