The X-linked mouse Rhox gene cluster contains more than 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. Here, we report the first identification and characterization of a Rhox5-regulated gene. This gene, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that we found is negatively regulated by Rhox5 in the testis in vivo. Transfection analyses in cell lines of different origin indicated that Rhox5-dependent down-regulation of Unc5c requires another Sertoli cell-specific cofactor. Examination of other mouse Rhox family members revealed that mouse RHOX2 and RHOX3 also have the ability to down-regulate Unc5c expression. The human RHOX protein PEPP2 (RHOXF2) also had this ability, indicating that Unc5c repression is a conserved RHOX-dependent response. Deletion analysis identified a Rhox5-responsive element in the Unc5c 5'-untranslated region. Although 5'-untranslated regions typically house post-transcriptional elements, several lines of evidence indicated that Rhox5 down-regulates Unc5c at the transcriptional level. The repression of Unc5c expression by Rhox5 may, in part, mediate the pro-survival function of Rhox5 in the testis, as we found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. Along with our other data, these findings led us to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival.