Cardinal Jose Saraiva Martins, head of the Vatican's sainthood in the church, led the mass.
"Today, we venerate his body, opening a particularly intense period of
pilgrimage,'' Saraiva Martins said.
"This body is here, but Padre Pio is not only a corpse. Looking at his remains we remember all the good that he has made.''
The Capuchin monk, who died in 1968, had an enormous following in Italy and abroad.
For decades he lived with inexplicable bleeding wounds on his hands and feet, like the wounds Jesus suffered at crucifixion.
Pope John Paul II made him a saint in 2002.
The body was exhumed by Church officials to allow faithfuls to pray before it to mark the 40th anniversary of his death.
The Church has taken measures to ensure that the body, which was unearthed in March, is being well preserved.
A team of medical examiners and biochemists worked to preserve and reconstruct the corpse.
The face has been covered by a silicone mask because it was apparently too decomposed to show.
After the unearthing the body was prepared for public viewing in the crypt of the Santa Maria delle Grazie church in San Giovanni Rotondo, a town near the Adriatic in southern Puglia.
Church officials said after an initial examination there was no sign of the so-called stigmata on his limbs, and the body was in good condition.
It is not yet known when the body will be reburied.
Padre Pio had a huge public following, with his beatification and canonization ceremonies drawing hundreds of thousands of people to the Vatican.
However, for decades many in the Vatican were uneasy about his popularity, doubting that his wounds were real and that his mystical virtues were authentic.
Despite his popularity he was banned from saying mass in public for many years.