Abstract Shiraia bambusicola can biosynthesize the valuable hypocrellin, which is applied in antibacterial, antitumoral, and antiviral fields. The lack of an effective transformation system limits studies on the hypocrellin metabolism deconstruction. We successfully expressed a lentiviral PgfpHyg vector in the Shiraia sp. SUPER-H168 by a polyethylene glycol and CaCl 2 transformation method. The transformant was resistant to 500 μg/mL of hygromycin B and represented by a green fluorescent signal in a fluorescent microscopy. We also overexpressed a multicopper oxidase (MCO) from the gene cluster of hypocrellin biosynthesis. MCO overexpression clearly enhanced hypocrellin production by about five times than the control strain, by improving the transcriptional levels of several hypocrellin metabolic genes. These genes included polyketide synthase, FAD/FMN-dependent oxidoreductase, monooxygenase, and O -methyltransferase. The increase of hypocrellin yield can efficiently solve the problem of its shortage from natural extraction. In summary, the polyethylene glycol and CaCl 2 regulation transformation was successfully applied in S. bambusicola with a lentiviral vector, which is mainly used in mammal cells. This study will lay a foundation for decoding hypocrellin metabolic pathway by further protein expression, bioconversion, and gene targeting. It will also create a new possibility for gene expression or further gene targeting of other wild filamentous fungi.