We have previously reported that Bmdsx, a homologue of the sex-determining gene doublesex ( dsx), was sex-specifically expressed in various tissues of the silkworm. The primary transcript of Bmdsx is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, we expressed BmDSXF in males from a ubiquitous promoter and examined its regulatory activities. We show that BmDSXF functions as a positive regulator of the hexameric storage protein termed SP1 and vitellogenin genes that are predominantly expressed in females. We also show that expression of Bmdsx(F) in males results in the repression of the pheromone-binding protein gene that is preferentially expressed in males. Gel-mobility shift assays demonstrated that BmDSX proteins bind to the sequence (ACATTGT) between -95 and -89 nt relative to the transcriptional initiation site of the vitellogenin gene. These results strongly suggest that Bmdsx is a final regulatory gene in the hierarchy of regulatory genes controlling the expression of female-specific protein in Bombyx mori.