The courtiers predict doom for Anna Bolena. Giovanna Seymour, one of her ladies-in-waiting has caught the eye of King Enrico VIII. Alone in her apartments, Anna asks the court musician, Smeton, to play for her. The sadness of his song upsets the queen (with whom he is in love) and she laments the emptiness of her life. 

Giovanna begins to suspect that Anna knows about her and the king. When the king enters she informs him she wishes to end their meetings. He tells he, that she will soon have no rival for his affections. Giovanna is adamant but the angry Enrico accuses her of loving the throne, not its occupant. She is consumed with remorse. 

The king sets about orchestrating Anna's ruin by arranging for her to come across her former lover, Lord Riccardo Percy, during a hunt. Percy confesses his love for Anna, but she demands that they do not see each other again. When Percy draws his sword to kill himself, Smeton misinterprets his actions and draws his own weapon. 

As Anna faints, Enrico bursts in and his wife is compromised. The king assumes the worst, and demands that Anna be tried.

Anna is imprisoned in the Tower. She prays for peace and solace. Giovanna appears and informs Anna that the king, and her successor, will spare her life if she confesses her guilt. When Giovanna admits that she is to be the new queen, Anna demands that she leave. Giovanna prostrates herself and begs for forgiveness. 

News arrives that Smeton has confessed to having had an affair with Anna, having been told that this would spare her life. Enrico bursts in and repeats Smeton's confession. Anna turns it back on the king. Giovanna confronts Enrico, and urges forgiveness and caution. 

In the Tower Anna recalls her childhood home, and the simplicity of her first love, and as she is summoned to the scaffold she sings a prayer. As cannon fire signals the marriage of Enrico and Giovanna, Anna calls on God to have mercy on the guilty couple.