The auction is scheduled for Oct. 27, said the COYS auction house.
The auctioneer said it expects the car to sell for several hundred thousand euros.
Cars that were said to have belonged to Pope Benedict XVI and his predecessor, Pope John Paul II, have also been auctioned off in the past. In 2005, a Volkswagen Golf fetched more than EUR188,000 on eBay after word emerged that it may have once belonged to Benedict.
The Mercedes-Benz was given to Padre Pio by a family grateful for a miracle that had been performed by the monk, said the auctioneer.
Padre Pio, who died in 1968, was considered the first priest in centuries to show signs of the stigmata, or the wounds suffered by Jesus at his crucifixion, on hands, feet and side. Countless faithful in Italy and around the world pray to him for miracles.
He was elevated to sainthood in 2002 by John Paul, himself a Padre Pio admirer.