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<i>Doublesex</i> (<i>Dsx</i>) is a polymorphic transcription factor of the <i>DMRTs</i> family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the <i>Dsx</i> gene is largely unknown in decapods. In this study, we reported the cDNA sequence of <i>PmDsx</i> in <i>Penaeus monodon</i>, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene <i>Dsx</i> was predominantly expressed in the <i>P. monodon</i> testis, and that <i>PmDsx</i> dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (<i>IAG</i>) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5′-flanking region of <i>PmIAG</i> that had two potential cis-regulatory elements (CREs) for the <i>PmDsx</i> transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that <i>PmDsx</i> overexpression significantly promoted the transcriptional activity of the <i>PmIAG</i> promoter via a specific CRE. These results suggest that <i>PmDsx</i> is engaged in male reproductive development and positively regulates the transcription of the <i>PmIAG</i> by specifically binding upstream of the promoter of the <i>PmIAG</i>. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in <i>P</i><i>. monodon</i>.