The ideal tumor antigen for the development of a cancer immunotherapy is one that is expressed only in tumor cells. The epidermal growth factor receptor pathway substrate 8 gene (Eps8) might be an effective antigen for cancer immunotherapy as it is overexpressed in a variety of cancer cells but not in normal tissues. In this study, the potential utility of an Eps8-derived immunotherapy was tested in vitro and in vivo. Three computer-based algorithms were used to design eight Eps8 native epitopes with potentially high binding affinity to the HLA-A2.1 molecule, which is found at a high frequency in the Chinese population. Of these eight, three peptides with a moderate affinity to the HLA-A2.1 molecule were modified at anchor residue positions to achieve stronger immunogenicity. These four modified peptides displayed stronger binding affinity to HLA-A2.1 molecules on T2 cells and a lower dissociation rate. In functional assays with human PBMCs in vitro and in HLA-A2.1/K(b) transgenic mice in vivo, CTLs primed by each native and modified peptide secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A2.1-restricted and Eps8-specific manner. p101-109-2L and p276-284-1Y9V were superior to other modified and native epitopes both in vitro and in vivo. These results indicate that employing the native and modified epitopes identified here in Eps8-based immunotherapy for HLA-A2.1 positive cancer patients may result in efficient anticancer immune responses for diverse tumor types.