Tumor-derived exosomes have been regarded as a new kind of cancer vaccine; however, their therapeutic efficacy needs to be further improved. Superantigen staphylococcal enterotoxin A (SEA)-coated tumor cells have been shown to potently induce tumor-specific T cell response. To increase efficacy of tumor-derived exosomes to induce antitumor immune response, we modified the exosomes by protein transfer of SEA tailed with a highly hydrophobic transmembrane domain (SEA-TM) and designated those SEA-TM-anchored exosomes as Exo/SEA-TM. We found the exosomes secreted from murine lymphoma E.G7-OVA cell line were round vesicles with the sizes of 40–100 nm limited by a bilayer membrane. Interestingly, the inner structure of the exosomes were visible under the transmission electron microscope; those “honeycomb-like” inner structure has not been described by other labs. Immunization with Exo/SEA-TM inhibited tumor growth and prolonged survival of the mice challenged with parental tumor cells more significantly than with exosomes (Exo) and even more than with the mixture of exosomes and SEA-TM. The results of mixed lymphocyte–tumor reaction (MLTR) showed that the increased IL-2, IFN-gamma secretion, and specific cytotoxic T lymphocyte (CTL) could be effectively induced from the splenic lymphocytes of the mice immunized with Exo/SEA-TM. In vivo depletion experiments showed that CD8⁺ T cells are the main effector cells, and both CD4⁺ T cells and NK cells are also involved in the antitumor effect of Exo/SEA-TM immunization. Therefore, tumor-derived exosomes surface anchored with SEA-TM can efficiently induce tumor-specific CTL thereby resulting in more potent inhibition of tumor growth. Our data provide an efficient and novel approach to tumor immunotherapy by protein modification of tumor-derived exosomes.