PF1163A (1) is a fungal metabolite that inhibits sterol-C4-methyl oxidase. In this study, we identified the biosynthetic gene cluster of 1 and elucidated its biosynthetic pathway through heterologous expression experiments. Polyketide synthase-nonribosomal synthetase hybrid PfaA, which is responsible for the biosynthesis of PF1163A, harbors an unusual domain organization with tandem condensation (C) domains and a terminal condensation domain. Mutagenesis studies suggest that both C domains are required for the function of PfaA.