Abstract Background Vaccination with tumor peptide epitopes associated with major histocompatibility complex class I molecules is an attractive approach directed at inducing tumor-specific cytotoxic T lymphocytes (CTLs). However, challenges remain in improving the therapeutic efficacy of peptide epitope vaccines, including the low immunogenicity of peptide epitopes and insufficient stimulation of innate immune components in vivo . To overcome this, we aimed to develop and test an innovative strategy that elicits potent CTL responses against tumor epitopes. The essential feature of this strategy is vaccination using tumor epitope-loaded nanoparticles (NPs) in combination with polyinosinic-polycytidylic acid (poly-IC) and anti-PD1 monoclonal antibody (mAb). Methods Carboxylated NPs were prepared using poly(lactic-co-glycolic acid) and poly(ethylene/maleic anhydride), covalently conjugated with anti-H-2KbmAbs, and then attached to H-2Kb molecules isolated from the tumor mass (H-2b). Native peptides associated with the H-2Kbmolecules of H-2Kb-attached NPs were exchanged with tumor peptide epitopes. The tumor-specific CTL-inducing and anti-tumor activities of the tumor epitope-loaded NPs were examined in mice bearing EG7-OVA thymoma or B16-F10 melanoma. In addition, the anti-tumor therapeutic efficacy of the NPs was examined in combination with poly-IC, anti-PD1 mAb, or both. Results Tumor peptide epitope-loaded NPs efficiently induced tumor-specific CTLs when used to immunize tumor-bearing mice as well as normal mice. This activity of the NPs significantly was increased when co-administered with poly-IC. Accordingly, the NPs exerted significant anti-tumor effects in mice implanted with EG7-OVA thymoma or B16-F10 melanoma, and the anti-tumor activity of the NPs was significantly increased when applied in combination with poly-IC. The most potent anti-tumor activity was observed when the NPs were co-administered with both poly-IC and anti-PD1 mAb. Conclusions Immunization with tumor epitope-loaded NPs in combination with poly-IC and anti-PD1 mAb in tumor-bearing mice can be a powerful means to induce tumor-specific CTLs with therapeutic anti-tumor activity.