Temperature-dependent sex determination is a notable model of phenotypic plasticity. In many reptiles, including the red-eared slider turtle Trachemys scripta elegans (T. scripta), the individual’s sex is determined by the ambient temperature during egg incubation. In this study, we show that the histone H3 lysine 27 (H3K27) demethylase KDM6B exhibits temperature-dependent sexually dimorphic expression in early T. scripta embryos before the gonad is distinct. Knockdown of Kdm6b at 26°C (a temperature at which all offspring develop into males) triggers male-to-female sex reversal in >80% of surviving embryos. KDM6B directly promotes the transcription of the male sex-determining gene Dmrt1 by eliminating the trimethylation of H3K27 near its promoter. Additionally, overexpression of Dmrt1 is sufficient to rescue the sex reversal induced by disruption of Kdm6b. This study establishes causality and a direct genetic link between epigenetic mechanisms and temperature-dependent sex determination in a turtle species.