The role of polyomavirus BK (BKV) large tumor antigen (L-Tag) as target of tumor immune surveillance in prostate cancer (PCa) patients has not been investigated so far. We thus analyzed the immunological profile exerted by L-Tag peptide-specific induction in newly diagnosed PCa patients, as compared to age-matched benign prostatic hyperplasia (BPH) patients, in relation to BKV L-Tag serology and urine molecular testing. We proposed here a comprehensive analysis of CTL response against BKV large T antigen-related epitopes in BKV-seropositive patients bearing prostate preneoplastic or neoplastic lesions irrespective of their HLA typing. We thus attempted the identification and characterization of antigen-specific immune responses against BKV L-Tag-peptides in either CD4+ or CD8+ T cells comparing functional features of peptide-expanded T cells in BKV seropositive PCa patients, aged-matched BPH, gendermatched healthy donors. Similarly, we investigated the efficiency of BKV L-Tag peptide-stimulated T cells to exert a cytotoxic activity against peptide-pulsed target cells across a wide range of HLA class I and II antigens.