Eukaryotic elongation factor 2 (eEF2) is an essential factor for protein synthesis. Previous studies have shown that the eEF2 gene was overexpressed and plays an oncogenic role in various types of cancers and that eEF2 gene product elicited both humoral immune responses to produce eEF2-specific IgG autoantibody in cancer-bearing individuals and cellular immune responses to induce eEF2 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro. The purpose of the present study was to induce eEF2-specific, antitumor CTL responses in vivo by vaccination with MHC class I-binding eEF2-derived peptide. First, two mouse MHC class I-restricted eEF2‑derived, 9-mer peptides, EF17 (17-25 aa, ANIRNMSVI) and EF180 (180-188 aa, RIVENVNVI) were identified as eEF2-specific CTL peptides, and mice were vaccinated intradermally eight times with either EF17 or EF180 peptide emulsified with Montanide ISA51 adjuvant. Cytotoxicity assay showed that eEF2-specific CTLs were induced in both EF17‑and EF180‑vaccinated mice, and histological study showed no detectable damage in the organs of these mice. Next, to examine in vivo antitumor effects of eEF2 peptide vaccination in a therapeutic model, mice were vaccinated four times with one each of the two eEF2 peptides at weekly intervals after implantation of eEF2-expressing leukemia cells. The vaccination with eEF2 peptides induced eEF2-specific CTLs and suppressed tumor growth, and disease-free survival was significantly longer in EF180-vaccinated mice compared to control mice. The survival was associated with the robustness of eEF2-specific CTL induction. These results indicate that vaccination with MHC class I-binding eEF2 peptide induced eEF2-targeting, antitumor CTL responses in vivo without damage to normal organs, which provided us a rationale for eEF2 peptide-based cancer immunotherapy.