In many reptiles, including the red-eared slider turtle Trachemys scripta elegans (T. scripta), sex is determined by ambient temperature during embryogenesis. We previously showed that the epigenetic regulator Kdm6b is elevated at the male-producing temperature and essential to activate the male pathway. In this work, we established a causal link between temperature and transcriptional regulation of Kdm6b. We show that signal transducer and activator of transcription 3 (STAT3) is phosphorylated at the warmer, female-producing temperature, binds the Kdm6b locus, and represses Kdm6b transcription, blocking the male pathway. Influx of Ca2+, a mediator of STAT3 phosphorylation, is elevated at the female temperature and acts as a temperature-sensitive regulator of STAT3 activation.