Scene 1: The Entrance to a Hidden Sanctuary
Armenia has been conquered by the Romans, and they have decreed that Christianity, which has a significant following in the country, must be destroyed and its followers put to death. Poliuto, the principal magistrate of Mytilene, has come to a secret gathering of worshippers to be baptised into the new faith. He confides to his friend Nearco, who is a fellow convert, that he has misgivings regarding his wife's loyalty to him. She was in love with a Roman general named Severo and only married Poliuto after pressure from her father, Felice, who told her that Severo and been killed in battle.
Poliuto enters the Sanctuary, and immediately his wife, Paolina, appears. She has followed him, suspecting that he has become a Christian convert. She waits for him to reappear from the baptism and, overhearing the service, finds herself strangely moved by its sincerity and power as the Christians pray for their persecutors. Nearco arrives with the news that not only is Severo still alive but that he intends to root out and destroy every Christian in the land. Paolina experiences both great joy and utter despair on learning that her lover has survived, but acknowledging that now they can never be united.
Scene 2: The Great Square of Mytilene
A jubilant crowd hails the arrival of Severo. He is overjoyed to see Paolina again, but his feelings quickly turn to rage and bitterness when he learns of her marriage to Poliuto.
Scene 1: The gardens of Felice's house
Severo angrily confronts Paolina. She tries to explain that she was tricked by her father and forced into marriage with Poliuto. Nevertheless she now intends to remain faithful to her husband and insists that Severo leaves her. Poliuto has learned of the meeting between the ex-lovers and is convinced of his wife's infidelity; however his bitter thoughts of revenge are interrupted by the news that Nearco has been arrested by the Romans for his religious beliefs.
Scene 2: The Temple of Jupiter
Nearco is dragged into the temple in chains. The priests demand to know the name of his important new convert to Christianity. When they threaten Nearco with torture, Poliuto proudly reveals himself as the man they seek. Paolina entreats her father to save her husband's life, and then throws herself at Severo's feet, begging him to show mercy for the sake of the love she knows he still has for her. Her actions so enrage Poliuto that he breaks free from his captors and smashes the pagan altar. He is quickly overpowered and led away with Nearco.
In his prison cell, Poliuto awakes from an uneasy sleep. He has had a dream that Paolina is in truth a loyal and faithful wife. She has persuaded the guards to let her visit him, and he forgives her and they are reconciled. Paolina urges him to save himself by renouncing his Christian beliefs, but he is certain that eternal salvation awaits him after death. Recognising the strength of his faith, Paolina begs him to baptize her, so that she can die with him. At first Poliuto is unwilling to perform the baptism, but when he sees that her conversion is genuine, he agrees.
Poliuto is to be fed to the lions, and Severo and his men arrive to take him to the arena. Severo is horrified when Paolina announces that she too is a Christian and demands to die with her husband. Severo urges her to reconsider, but she remains determined to join Poliuto in martyrdom. They are led away together to face their gruesome fate.