Chinese soft-shelled turtles (Pelodiscus sinensis) possess apparent sexual dimorphism on production traits, and the realization of all-male breeding is the important measure for the improvement of the industry. In this study, the highly conserved gene sox9 in vertebrate male differentiation was chosen as manipulating object, and by injecting Lentivirus-sox9-shRNA interference and overexpression vector system into embryos before sexual differentiation, we discussed the specific function of sox9 during the early testicular differentiation of P. sinensis from the level of histology and molecular study. The results of the loss-of-function experiments showed that the appearance and internal structure of ZZ embryonic gonads with sox9 knockdown underwent significant feminization, and the germ cells presented female distribution pattern. The mRNA expression of male specific gene dmrt1 and amh significantly decreased, and that of female specific gene foxl2 obviously increased. Besides, the expressive signals of the DMRT1 protein almost disappeared, while FOXL2 protein was activated and abundantly expressed, indicating complete sex reversal from male to female on gonads(ratio of reversal: 87.9%). The results of the gain-of-function experiments showed that 42.5% of ZW embryos overexpressing sox9 differentiated towards male. The expression of dmrt1 and amh were upregulated while foxl2 was downregulated, and the fluorescent signals of both DMRT1 and FOXL2 were detected simultaneously in the same gonad, indicating incomplete sex reversal. These findings above demonstrate that sox9 is the necessary gene for early testicular formation in P. sinensis, and it does participate in the process of regulating male differentiation, which establishes theoretical foundation for the following analysis of sexual differentiation mechanism of P. sinensis and provides breeding target for realizing the sex control of P. sinensis at the same time.