Abstract: To explore the function of the sox9a gene in gonadal differentiation of yellow drum and its regulatory effect on the dmrt1a gene, we cloned the CDS sequence of sox9a and analyzed its molecular characteristics and regulatory functions. The results of qRT-PCR detection showed there was no significant difference in its expression between male and female during the critical period of sex determination (36 dph), while at 55 days post hatching (dph) and 61 dph, the expression of sox9a in males was significantly higher than that in female (P<0.01). Molecular docking and molecular dynamics simulation suggested that Sox9a might regulate the expression of dmrt1a. Furthermore, the dual-luciferase reporter assay system and site-directed mutagenesis technology were employed to detect the regulatory role of Sox9a on dmrt1a gene expression in CHO and 293T cells. The results demonstrated that Sox9a could enhance the transcriptional activity of dmrt1a_Luc, and its promoting effect on dmrt1a_Luc located on the Y chromosome was stronger than that on the X chromosome; with the increase in Sox9a concentration, its ability to promote the transcriptional expression of dmrt1a_Luc decreased accordingly. Mutation of the Sox9a binding site within the 45 bp insertion fragment in Intron 1 of dmrt1a gene on the X chromosome resulted in significantly higher transcriptional activity of the mutant Pro_Intron1 (X_Mu) construct compared to the wild type, indicating this Sox9a binding site plays an important role in the transcriptional regulation of dmrt1a. This study enhances the understanding of the role of Sox9a in testicular differentiation of yellow drum and provides important insights for elucidating the molecular mechanisms underlying sex determination and differentiation in this species.