Abstract: The molecular mechanism of sex determination in Pelodiscus sinensis has been a long-standing mystery. In many vertebrate species, estrogens play a key role in sex determination and gonadal differentiation. To investigate the effect of estrogens in the gonadal differentiation of P. sinensis and the underlying molecular mechanism, we treated the undifferentiated embryos with 17-estradiol (E2) and aromatase inhibitor (AI). Results showed that the female ratio increased by 92.3% after E2 treatment, whereas female ratio in AI treatment group decreased dramatically to 13.1%. Meanwhile, the obvious masculinization of genetically female embryos and feminization of genetically male embryos were observed after AI and E2 treatment, respectively. Moreover, RT-PCR and immunofluorescence analysis showed that E2 could significantly downregulated mRNA and protein expressions of DMRT1 and SOX9, which were the key factors involving in male development. However, AI exerted the opposite effect. In conclusion, estrogens inhibited the male development via the inhibition of expression of DMRT1 and SOX9, and consequently led to female development, indicating the important role of estrogens in the female sexual differentiation in Pelodiscus sinensis.