In the silkworm, the sex-determination primary signal Fem controls sex differentiation by specific binding of Fem -derived piRNA to the cleavage site in Masc mRNA, thus inhibiting Masc protein production in the female. In this study, we identified a novel splicing isoform of Masc , named Masc-S , which lacks the intact sequence of the cleavage site, encoding a C-terminal truncated protein. Results of RT-PCR showed that Masc-S was expressed in both sexes. Over-expression of Masc-S and Masc in female-specific cell lines showed that Masc-S could be translated against the Fem -piRNA cut. By RNA-protein pull-down, LC/MS/MS, and EMSA, we identified a protein BmEXU that specifically binds to an exclusive RNA sequence in Masc compared to Masc-S . Knockdown of Masc-S resulted in abnormal morphology in female external genital and increased expression of the Hox gene Abd-B , which similarly occurred by Bmexu RNAi. These results suggest that the splice variant Masc-S against Fem -piRNA plays an important role in female external genital development, of which function is opposite to that of full-length Masc . Our study provides new insights into the regulatory mechanism of sex determination in the silkworm.