The Rhox cluster on the mouse X chromosome contains reproduction-related homeobox genes expressed in a sexually dimorphic manner. We report that two members of the Rhox cluster, Rhox6 and 9, are regulated by de-methylation of histone H3 at lysine 27 by KDM6A, a histone demethylase with female-biased expression. Consistent with other homeobox genes, Rhox6 and 9 are in bivalent domains prior to embryonic stem cell differentiation and thus poised for activation. In female mouse ES cells, KDM6A is specifically recruited to Rhox6 and 9 for gene activation, a process inhibited by Kdm6a knockdown in a dose-dependent manner. In contrast, KDM6A occupancy at Rhox6 and 9 is low in male ES cells and knockdown has no effect on expression. In mouse ovary where Rhox6 and 9 remain highly expressed, KDM6A occupancy strongly correlates with expression. Our study implicates Kdm6a, a gene that escapes X inactivation, in the regulation of genes important in reproduction, suggesting that KDM6A may play a role in the etiology of developmental and reproduction-related effects of X chromosome anomalies.