[Objective] Coprisidins (A and B) are structural unique naphthoquinone-oxindole alkaloids from an insect gut-associated Streptomyces with bioactivities for cancer prevention. As the first example of natural alkaloids with naphthoquinone-oxindole skeleton, the biosynthesis of coprisidins remains unclear. Exploration of the coprisidins biosynthetic mechanism would gain new insights into the biosynthesis of type II polyketide natural products. [Methods] The genome of coprisidin-producing strain Streptomyces sp. SNU607 was sequenced and in silico analysis revealed the biosynthetic potential. The coprisidins biosynthetic cluster was cloned and identified by the gene inactivation and heterologous expression experiments. A biosynthetic pathway for coprisidins was proposed according to the in vivo results and bioinformatics analysis. [Results] Genome analysis of Streptomyces sp. SNU607 revealed 23 gene clusters potentially for secondary metabolites with four of them related to polyketide synthases (PKSs). Gene inactivation and heterologous expression confirmed that a type II PKS gene cluster, which contains 30 putative open reading frames, is responsible for coprisidins biosynthesis. The copH/I/M/N/O genes could constitute a five gene cassette for the biosynthesis of butyryl start unit of coprisidins. Sequence alignment of KS_β (CopB) suggested that coprisidins are biosynthesized by a type II system with a nascent chain length of C20, which is afterward modified by a variety of accessory enzymes. [Conclusion] The rare naphthoquinone-oxindole backbone of coprisidins is assembled by the minimal type II PKS (CopB-C-A) utilizing a butyryl starter unit, along with ketoreductases and cyclases to form the anthracycline-like four ring system. Subsequently, tailoring enzymes and oxidative arrangement are proposed to shape the final naphthoquinone-oxindole skeleton. Considering the structure feature of coprisidins, this study would set the stage for biosynthetic mechanism studies of naphthoquinone-oxindole alkaloids and expand the structural diversity of products synthesized by type II PKSs.