Citreoviridin (1) belongs to a class of F1-ATPase beta-subunit inhibitors that are synthesized by highly reducing polyketide synthases. These potent mycotoxins share an alpha-pyrone polyene structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution using heterologous expression in Aspergillus nidulans are reported. Two intermediates were isolated that allowed the proposal of the biosynthetic pathway of citreoviridin.