The highly conserved doublesex (dsx) and doublesex/mab-3 related (Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, DsxM in males and DsxF in females, which function differentially and often oppositely to establish sexual dimorphism. Here, we report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane (BM) protein Collagen IV in the fat body. We find that Dsx isoforms regulate the expression of Collagen IV in the fat body and its secretion into the BM of other tissues. We identify the procollagen lysyl hydroxylase (dPlod) gene, which is involved in the biosynthesis of Collagen IV, as a direct target of Dsx. We further show that Dsx regulates Collagen IV through dPlod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen IV and remotely establish sexual dimorphism.