Abstract: The regulatory role of Amh in early sex determination and gonadal differentiation has been studied in some GSD (genetic sex determination) animals, such as fish, however, the biological function of this gene in species with temperature-dependent sex determination (TSD) has not been reported. In this study, the red-eared slider turtle Trachemys scripta was used as an animal model of TSD to analyze the expression characteristics and cellular localization of Amh in embryonic gonads. Then, the specific function of Amh in TSD was identified by means of gene loss- and gain-of-function studies (LV-Amh-shRNA and LV-Amh-OE vector systems) . The expression analysis showed that Amh was highly expressed in MPT (male-producing temperature) gonads of stage 15, prior to the onset of gonadal differentiation. AMH protein was mainly localized in the precursor sertoli cells of MPT gonads, however, the FPT (female-producing temperature) gonads exhibited extremely low expression levels of Amh mRNA and protein throughout the embryogenesis. RNA interference experiments showed complete male-to-female sex reversal in MPT gonads with Amh knockdown, characterized by obvious feminization of gonads, female-styled germ cell distribution, significant downregulation of testicular marker Sox9, and remarkable upregulation of ovarian markers Foxl2 and Cyp19a1. Conversely, ectopic expression of Amh led to largely masculinized FPT gonads, a decline in Foxl2 and Cyp19a1, and production of Sox9. The statistical results showed that the sex reversal rates induced by LV-Amh-shRNA and LV-Amh-OE were 92.86% (26/28) and 66.67% (26/39), respectively. These data provided evidence that Amh is both necessary and sufficient to initiate early testicular differentiation in T. scripta, and it is located upstream of the male molecular pathway of TSD, laying the foundation for the analysis of the TSD molecular mechanism.