Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex deter-mination cascade in the silkworm,Bombyx mori,provides novel insights into high di-versity of insect sex determination pathways.In this system,the W-chromosome-derived Fem piRNA is the primary sex determination signal.A CCCH-type zinc finger gene Mas-culinizer (Masc),which is targeted by Fem piRNA-PIWI complex in female animals,is indispensable for male-specific splicing of B.mori doublesex (Bmdsx).Although many genes involved in this cascade have been identified,the regulatory mechanisms of silk-worm sex determination remain to be elucidated.Here we show that another CCCH-type zinc finger gene,Bmznf-2,is a masculinization factor in B.mori.Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized dif-ferentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males.In contrast,there is no phenotypic consequence in mutant females.In mutant males,relative messenger RNA expression levels of female-dominant genes such as vitel-logenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated.Furthermore,male mutants show de-layed developmental timing,smaller body sizes of larvae and malformation of moth wings.Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation.