Abstract: The molecular mechanisms underpinning temperature-dependent sex determination (TSD) in reptiles have attracted great biological interest for many years. However, which genetic factors are essential for TSD remain elusive, especially regarding female sex determination. Cytochrome P450 family 19 subfamily A member 1 (Cyp19a1) encodes the endoplasmic reticulum enzyme aromatase, which participates in the catalytic conversion of androgens to estrogens, and is implicated in sexual differentiation in many species. However, whether Cyp19a1 plays a critical role in determining gonadal sexual fate in TSD species remains to be elucidated. In the current study, Cyp19a1 exhibited a temperature-dependent and sexually dimorphic expression pattern, preceding gonadal sex differentiation in a TSD turtle species (Mauremys reevesii). Sexual phenotype of the turtles was successfully reversed by aromatase inhibitor treatment at the female-producing temperature (FPT). Furthermore, exogenous estradiol (E2) treatment led to complete male-to-female sex reversal at the male-producing temperature (MPT), accompanied by rapid up-regulation of Cyp19a1. Thus, Cyp19a1 appears to be essential for female sex determination in M. reevesii, suggesting a vital role in the female TSD pathway.