The major setback in natural product screening is the decreasing hit rate of novel bioactive compounds containing new chemical skeletons. Here we report the identification and biosynthesis of isoindolinomycin (Idm), an unprecedented bioactive polyketide with a novel isoindolinone-containing tetracyclic skeleton. Idm was discovered through the screening of rifampicin-resistant ( rif) mutants that were generated from nine actinomycete strains used in this study. Of the 114 rif mutants isolated, the mutant S55-50-5 was found to overproduce Idm, which is almost undetectable in the wild-type Streptomyces sp. SoC090715LN-16. An in silico analysis coupled with gene deletion experiments revealed a biosynthetic idmB gene cluster that is responsible for the production of Idm. The biosynthetic studies of Idm primarily focused on the formation of the five-membered ring in the tetracyclic structure and the attachment of the methyl group to the core structure. In addition, a malachite green phosphate assay performed using a stand-alone adenylation domain ( idmB21) demonstrated the involvement of glycine in the formation of the isoindolinone-containing skeleton. This study contributes to an increase in the structural diversity of polyketides and paves the way toward an understanding of the complete biosynthetic pathway of a novel class of tetracyclic polyketides.