A small group of believers carried a cross between 14 "stations" evoking the last hours of Jesus's life during the traditional Via Crucis (Way of the Cross) procession, as the Pope looked on.
Tens of thousands gathered for the service, many clutching candles in the imposing surrounds of the Colosseum.
"Lord Jesus, inwardly support the persecuted. May the fundamental right to religious freedom spread far and wide," believers heard in a "meditation" written by Italian bishop Renato Corti.
"Men and women are imprisoned and even killed solely because they are believers or committed to justice and peace. They are not ashamed of the cross. They are outstanding examples to everyone," Corti's meditation continued.
The procession was led by believers from Iraq, Syria and Egypt -- all of whose minority Christian populations face persecution from armed groups including Islamic State militants -- and Nigeria, where thousands have been murdered by jihadist organisation Boko Haram.
Attacks in Kenya
The focus on religious persecution came after Thursday's day-long siege of Garissa University, Kenya's deadliest attack since the 1998 bombing of the US embassy in Nairobi, and the bloodiest ever by the Al-Qaeda-affiliated militants.
Hurling grenades and firing automatic rifles, Shebab gunmen stormed the university at dawn as students were sleeping, shooting dead dozens before setting Muslims free and holding Christians and others hostage.
"In union with all people of good will throughout the world, His Holiness condemns this act of senseless brutality and prays for a change of heart among its perpetrators," a papal telegram sent earlier on Friday said.
Corti's meditation also contained a passage expressing hope for an end to the death penalty, a long-held position of the Church, and went on to describe paedophilia as "barbaric".
"You have given us immense dignity, you beckon us towards freedom. Free us from all forms of slavery," the meditation said, in prayer for child soldiers and victims of human trafficking.