Significance Testosterone has been implicated as a regulator of the immune response to viruses, vaccines, host tissue, and cancer. Despite this pleiotropic effect on the immune system, the mechanisms underlying this effect are not well understood. In this study, we investigated how testosterone altered gene expression and signaling mechanisms in CD4 T cells in mouse models and prostate cancer patients undergoing androgen deprivation therapy. We found that testosterone inhibited T-helper 1 differentiation by up-regulating the phosphatase, Ptpn1 , in both mice and humans. Additionally, the androgen receptor bound a highly conserved region of the Ptpn1 gene, suggesting an evolutionarily important purpose of this mechanism. This study provides a mechanism to explain recent discoveries regarding the role of testosterone-mediated inhibition of the immune response.