The human leukocyte antigen (HLA)-G molecule can exert immunoregulatory functions. However, its limited tissue distribution and preferential expression in a variety of malignancies suggest the possibility that it could be a target in anti-cancer immunotherapy. In the present study, we tested this possibility by focusing on renal cell carcinoma (RCC) patients, especially those with HLA-A24 alleles. Four HLA-G-derived peptides were prepared based on the binding motif to the HLA-A24 alleles. After a stabilization assay confirmed the binding of these peptides to HLA-A24 molecules, they were screened for the capacity to induce peptide-specific cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) of HLA-A24+ RCC cancer patients. As a result, the HLA-G(146-154) peptide was found to effectively induce peptide-specific CTLs, and HLA-G(146-154) peptide-stimulated PBMCs exhibited cytotoxic activity against HLA-G-expressing HLA-A24+ RCC cells. Antibody blocking and cold inhibition experiments confirmed that the cytotoxicity was partially ascribed to peptide-specific and HLA class I-restricted CD8+ T cells. These results indicate that HLA-G-associated immunoregulation can be overcome and that HLA-G peptide-based anti-cancer immunotherapy is feasible.