The relics of the saint are on display at Rome's San Lorenzo Basilica, as part of the Roman Catholic Church's special jubilee celebrations.
St. Leopold Mandic's remains are also on display at the same church.
They were unveiled following a solemn procession in Via della Conciliazione and on Friday will reside in St Peter's Basilica until 11 February.
The Vatican said the relics would be displayed before the Altar of the Confession.
On Thursday morning, hundreds of people gathered to view the remains with some breaking into tears whilst others prayed silently.
Padre Pio is often referred to as one of the Catholic Church's most popular saints.
After living his life as a monk, he died in 1968 with the "stigmata" - the bleeding wounds left on the hands and feet of Jesus following his crucifixion.
His body was originally exhumed in 2008 and has been partially reconstructed with a lifelike silicone mask and is being stored in a temperature-controlled glass box.
Despite several allegations and criticism that Padre Pio was a fake Church officials cleared him each time.
He was canonised in June 2002 by Pope John Paul II.
His feast day is celebrated on 23 September and he is patron saint of civil defence volunteers, adolescents, Pietrelcina, Stress relief and the January blues.
