Hypoxic tumor microenvironment makes cancer cells to be therapy-resistant and hypoxia-inducing factors (HIFs) play a central role in hypoxic adaptation. Especially, renal cell carcinoma (RCC) is often associated with von Hippel-Lindau (VHL) gene mutations, leading to up-regulation of HIFs. However, from a different point of view, this suggests the possibility that HIFs could be promising targets in anti-cancer therapy. In this study, we searched for HIF-1α-derived peptides that are able to induce RCC-reactive cytotoxic T lymphocytes (CTLs) from HLA-A24+ RCC patients. Among five peptides derived from HIF-1α, which were prepared based on the binding motif to the HLA-A24 allele, a HIF-1α278–287 peptide induced peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24+ RCC patients most effectively. In immunoblot assays, the expression of HIF-1α was lowly detected in whole and nuclear lysates of RCC cell lines even under normoxia (20% O2), and their expression in whole lysates was increased under hypoxia (1% O2). Additionally, HIF-1α278–287 peptide-stimulated T cells showed a higher cytotoxicity against HLA-A24+ HIF-1α-expressing RCC cells than against HLA-A24− HIF-1α-expressing RCC cells. The cytotoxicity was inhibited by the addition of HIF-1α278–287 peptide-pulsed cold target cells. Altogether, these results indicate that the HIF-1α278–287 peptide could be a candidate for peptide-based anti-cancer vaccines for HLA-A24+ RCC patients.