Background: CD8(+) cytotoxic T lymphocytes (CTLs) have been proved to exert crucial roles in immunological rejection. Exosomes (EXOs) secreted by CD4(+)CD25(+) regulatory T (Treg) cells is believed to be deeply involved in immune regulation. Nevertheless, whether immunomodulatory effect of CD4(+)CD25(+) Treg cells on CD8(+) CTL depends on EXOs remains unknown and needs to be explored. Material/Methods: We purified CD4(+)CD25(+) Treg cells followed by in vitro amplification. EXOs in culture supernatants of Treg cells was separated and identified. The effect of CD4(+)CD25(+) Treg cells and CD4(+)CD25(+) Treg cells-derived EXOs on CD8(+) CTL viability, proliferation, cell cycle mRNA, intracellular cytokines, and protein expression were investigated. Results: We successfully obtained EXOs from CD4(+)CD25(+) Treg cells. The inhibition effect of EXOs on CD8(+) CTL was concentration-dependent. In addition, the inhibition effect of CD4(+)CD25(+) Treg cells could be reversed by GW4869, an EXOs inhibitor. The inhibition effect was associated with the regulation of IFN-gamma and perforin. Our in vivo experiments proved that natural CD4(+)CD25(+) Treg cells-released EXOs can prolong liver allograft survival. Conclusions: CD4(+)CD25(+) Treg cells-derived EXOs could become an alternative tool for manipulating the immune system to discover novel underlying immunomodulatory mechanisms.