Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxicT lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loadedwith TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bonemarrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loadedwith either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flowcytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8,colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-β and IL-12were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loadedDCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activatedby DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2cells in vitro. The secretion levels of IFN-γ, TNF-β and IL-12, secreted by DCs loaded with antigen and PEP-3and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activatedby DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiencyagainst TRAMP-C2 cells in vitro.