In reptiles with temperature-dependent sex determination(TSD), the individual’s gender depends completely on the incubation temperature of the eggs. The molecular mechanism underlying temperature- dependent sex determination has been a long-standing mystery, in particular, the factors responsible for triggering the differentiation of biopotential gonads either into a testis or an ovary are unknown. In this study, the regulatory role of Sox9 on male gonadal differentiation in Trachemys scripta was investigated through expression analysis and gene-modulating approach, providing important reference for the study of sexual differentiation and gonadal development mechanism of reptiles with TSD and an effective molecular techniques for controlling the sex of animals. Real-time PCR showed that the MPT-specific expression of Sox9 in embryonic gonads initiated early at stage 17, preceding the onset of gonadal differentiation, however, the FPT embryonic gonads exhibited extremely low expression level of Sox9 throughout the embryogenesis. Immunofluorescence further confirmed the SOX9 protein was abundantly expressed and mainly localized in the nuclei of precursor Sertoli cells in MPT gonads. The temperature shifts experiments showed that the expression of Sox9 in MPT→FPT (which was shifted at stage 16) gonads was significantly reduced from the stage 17, indicating that Sox9 can respond rapidly to the new temperature. Meanwhile, the expression of Sox9 was also down-regulated rapidly in the MPT gonads treated with estrogen. Loss-of-function analysis showed that 90.9% MPT embryos with Sox9 knockdown exhibited male to female sex reversal, characterized by obvious feminization gonads with well organized outer cortex and degradated medullary area. These above results indicate that Sox9 is required for testicular differentiation in a TSD system.