During the song Live To Tell, she stands Jesus Christ-like on a huge cross, wearing a fake crown of thorns. And it is that image that has outraged senior Roman Catholics.
In a show of religious solidarity, Muslim and Jewish leaders have also condemned the singer, who was brought up as a Catholic and is famous for peppering her concerts and videos with religious and sexual imagery.
Madonna's "Confessions" tour reaches Rome on Sunday - it is a 75,000 sell-out - and one senior figure close to Pope Benedict XVI is calling for her to be excommunicated.
Cardinal Ersilio Tonino, speaking with the Pope's approval, said: "This time the limits have really been pushed too far. This [concert] is a blasphemous challenge to the faith and a profanation of the cross. She should be excommunicated.
"To crucify herself during the concert in the city of popes and martyrs is an act of open hostility. It is nothing short of a scandal. What is really offensive is the exaggeration of it all; it is clearly anti-Church, in poor taste and, most of all, an insult to Christ.
"The time will come when this woman will realise that Christ died on the cross for her as well, that He spilt His blood for her - I feel pity for her."
Another Vatican official, Bishop Velasio De Paolis, said: "Wizards and satanists use religious objects for black masses and she is simply following them. How this woman can take the name of the mother of Christ I don't know. Her show represents the rotten fruit of secularism and the absurdity of evil."
Mario Scialoja, the head of Italy's Muslim League, said. "I think her idea is in the worst taste and she'd do better to go home."
Riccardo Pacifici, vice-president of the Roman Jewish community, said Madonna should have pulled the routine given that she was performing in a stadium only a mile from the gates of Vatican City.
It is not the first time the star has raised the ire of the Catholic church. Its leaders condemned as blasphemous her 1989 Like a Prayer video, featuring burning crosses, statues crying blood and Madonna seducing a black Jesus.
The 47-year-old singer has refused to kowtow to Italy's Catholic sensibilities, but in an interview published on Thursday, she had nothing but praise for her Italian background.
She told the local edition of Vanity Fair that her "good Italian genes" were the reason for her staying in shape, famously allowing her to writhe in a leotard in a recent video.
Madonna has insisted that her dressing room at the Olympic Stadium on Sunday be decorated all in white, complete with white roses and Japanese soya snacks.