HER2/neu is one of the most appropriate target antigens for anti-cancer therapy because of its expression in various types of epithelial cancer. HER2/neu can also be a target for both cellular and humoral immune responses. In this study, we attempted to identify HER2/neu-derived peptides, which are able to be recognized by both humoral and cellular immune systems in HLA-A2+ cancer patients. Among 12 HER2/neu-derived peptides having the HLA-A2 binding motifs, immunoglobulin G reactive to each of 7 HER2/neu peptides was detected in the plasma of >50% of cancer patients. Among these 7 peptides, 3 including HER2/neu 444-452, HER2/neu 466-474, and HER2/neu 484-492, effectively induced peptide-specific and HLA-A2-restricted cytotoxic T lymphocyte activity from peripheral blood mononuclear cells of cancer patients, regardless of different HLA-A2 subtypes. Experiments using blocking antibodies and cold inhibition targets revealed that the cytotoxicity against HER2/neu-expressing HLA-A2+ tumor cells was peptide-specific and CD8+ T cell-dependent. Overall, these results indicate that these 3 HER2/neu-derived peptides are efficiently recognized by both the humoral and cellular immune systems, and therefore could be useful for peptide-based immunotherapy for HLA-A2+ patients with various types of epithelial cancer.