Azinomycin B is a complex natural product containing densely assembled functionalities with potent antitumor activity. Cloning and sequence analysis of the azi gene cluster revealed an iterative type I polyketide synthase (PKS) gene, five nonribosomal peptide synthetases (NRPSs) genes and numerous genes encoding the biosynthesis of unusual building blocks and tailoring steps for azinomycin B production. Characterization of AziB as a 5-methyl-naphthoic acid (NPA) synthase showed a distinct selective reduction pattern in aromatic polyketide biosynthesis governed by bacterial iterative type I PKSs. Heterologous expression established the PKS-post modification route from 5-methyl-NPA to reach the first building block 3-methoxy-5-methyl-NPA. This proposed azinomycin B biosynthetic pathway sets the stage to investigate the enzymatic mechanisms for building structurally unique and pharmaceutically important groups, including the unprecedented azabicyclic ring system and highly active epoxide moiety.