Objective: To induce allogeneic antigen-specific CD4+CD25+ regulatory T cells in vivo and identify the function of these cells. Methods: Allogeneic antigen-specific CD4+CD25+ regulatory T cells were generated by the addition of multiple intravenous injections of ICR mice splenocytes in vivo. Spleen cells were then pooled and enriched for CD4+CD25+ T cells and CD4+CD25- T cells by negative and positive selection using MACS in vitro. Characterizations of allogeneic antigen-specific CD4+CD25+ regulatory T cells were tested by mixed lymphocyte reaction(MLR) consisting of BALB/cByJ splenocytes as responder and MMC-treated ICR splenocytes as stimulator. The abilities of these cells to respond to various stimulators and to suppress alloreactive T cells responses were tested in vitro. Results: In the MLR, allogeneic antigen-specific T cells displayed little proliferation with allogeneic-antigen existence. Furthermore, these cells inhibited the proliferative responses and IFN-γ production of na-觙ve CD4+CD25- T cells to alloantigens in vitro. Conclusion: Donor-specific splenocyte transfusion-induced CD4+CD25+ T cells were allogeneic antigen-specific. They were anergic,and retained their ability to suppress antigen-driven responses of CD4+CD25- T cells in vitro.