<jats:title>Abstract</jats:title><jats:p>Himeic acid A, which is produced by the marine fungus <jats:italic>Aspergillus japonicus</jats:italic> MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by <jats:italic>Agrobacterium</jats:italic>‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the <jats:italic>him</jats:italic> biosynthetic gene cluster for himeic acid in <jats:italic>A. japonicus</jats:italic> MF275 has been identified.