The Dmrt gene family, with conserved functions throughout evolution, contains DM domains that play roles in various physiological processes like sex determination and gonadal differentiation. The mud crab (Scylla paramamosain), a significant marine culture crab in China, exhibits sexual dimorphism and is an important subject for reproduction research. In this study, the ORFs of the SpDmrt-1 and SpDmrt-3 genes from the mud crab gonad transcriptome database were obtained and confirmed by PCR. Both genes were expressed at the highest level in the testis among various tissues and in the T2 stage testis during different gonad developmental stages. In situ hybridization results showed that both genes were expressed during spermatogenesis. Silencing one gene led to a decrease in the expression of the other. Moreover, the expressions of some gonadal development-related genes (dsx, foxl-2, vasa, Dmrt-like, and IAG) changed significantly. Transcriptomic data indicated that SpDmrt-1 and SpDmrt-3 regulate various signaling pathways, including the Notch signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, the Calcium signaling pathway, and several other gender-related pathways, showing their involvement in male mud crab development. Furthermore, after long-term interference with SpDmrt-1 in vitro, most cells in the testis of the mud crab were at the spermatid stage, only a small amount of spermatozoa could be observed, and obvious empty plaque-like areas appeared in the tissue. Our findings provided a crucial foundation for understanding the role of the Dmrt in the process of gonadal development in crustaceans.