Discovery of immunologically relevant antigens in prostate cancer forms the basis for developing more potent active immunotherapy. We report here a strategy using the transgenic adenocarcinoma of mouse prostate (TRAMP) model, which allows for the functional identification of immunogenic prostate tumor antigens with relevance for human immunotherapy. Using a combination of active tumor vaccination in the presence of CTL-associated antigen 4 (CTLA-4) in vivo blockade, we elicited tumor-specific T cells used to expression clone the first T cell-defined TRAMP tumor antigen, called Spas-1 (stimulator of prostatic adenocarcinoma specific T cells-1). Spas-1 expression was increased in advanced primary TRAMP tumors. We show that the immunodominant SPAS-1 epitope SNC9-H(8) arose from a point mutation in one allele of the gene in TRAMP tumor cells, and that immunization with dendritic cells pulsed with SNC9-H(8) peptide resulted in protection against TRAMP-C2 tumor challenge. In humans, the Spas-1 ortholog SH3GLB2 has been reported to be overexpressed in prostate cancer metastases. Additionally, we identified a nonmutated HLA-A2-binding epitope in the human ortholog SH3GLB2, which primed T cells from healthy HLA-A2(+) individuals in vitro. Importantly, in vitro-primed T cells also recognized naturally processed and presented SH3GLB2. Our findings demonstrate that our in vivo CTLA-4 blockade-based T cell expression cloning can identify immunogenic cancer antigens with potential relevance for human immunotherapy.