Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady state amount, compared with cytosolic-localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.