Abstract Although androgen ablation therapy is effective in treating primary prostate cancers, a significant number of patients develop incurable castration-resistant disease. Recent studies have suggested a potential synergy between vaccination and androgen ablation, yet the enhanced T-cell function is transient. Using a defined tumor antigen model, UV-8101-RE, we found that concomitant castration significantly increased the frequency and function of antigen-specific CD8+ T cells early after the immunization of wild-type mice. However, at a late time point after immunization, effector function was reduced to the same level as noncastrated mice and was accompanied by a concomitant amplification in CD4+CD25+Foxp3+ regulatory T cells (Treg) following immunization. We investigated whether Treg expansion occurred following castration of prostate tumor–bearing mice. In the prostate-specific Pten−/− mouse model of prostate cancer, we observed an accelerated Treg expansion in mice bearing the castration-resistant endogenous prostate tumor, which prevented effector responses to UV-8101-RE. Treg depletion together with castration elicited a strong CD8+ T-cell response to UV-8101-RE in Pten−/− mice and rescued effector function in castrated and immunized wild-type mice. In addition, Treg expansion in Pten−/− mice was prevented by in vivo interleukin (IL)-2 blockade suggesting that increased IL-2 generated by castration and immunization promotes Treg expansion. Our findings therefore suggest that although effector responses are augmented by castration, the concomitant expansion of Tregs is one mechanism responsible for only transient immune potentiation after androgen ablation. Cancer Res; 72(8); 1975–85. ©2012 AACR.