AbstractBACKGROUNDGenetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P‐element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male‐specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown.RESULTSHere we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR–Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male‐specific defects in internal and external genitals. In addition, we detected female‐specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex‐biased genes and induced male sterility.CONCLUSIONOur study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic‐based pest management in lepidopteran pests. © 2021 Society of Chemical Industry