Previous data have demonstrated that mesenchymal stem cells (MSCs) can exert immunomodulatory activity in vitro, in which of the process nearly all kinds of immune cell subsets are involved. However, there is still a paucity of information about whether and why MSCs inhibit the ongoing immune responses in vivo. Working in a murine splenocyte transfusion model across the major histocompatibility barrier (C57BL/6 → BALB/c, H2 b → H2 d ), we have found that MSC coinfusion prolongs the mean survival time (MST) of the recipient mice in a dose-dependent manner and reduces graft-versus-host-associated histopathology in comparison to the allosplenocyte transfusion controls. In vivo eGFP tracing with polymerase chain reaction analysis revealed that grafted MSCs could migrate and settle into the lungs, spleen, liver, intestine, and skin shortly after administration. Further investigations into the functional characteristics of intrasplenic lymphocytes showed that their proliferation and cytotoxic activity against P815 cells (H2 d ) were significantly restrained by MSC cotransfer. FACS analysis demonstrated that MSC infusion not only increased the proportion of CD4 + subset but also decreased that of CD8 + cells at the belated observation points, resulting in the increase of the ratio of CD4 + /CD8 + cells. Also, in contrast to the slight increase of the proportion of CD4 + CD25 + T regulatory cells (Tregs) in MSC cotransfer mice, the ratio of Tregs/CD8 + cells was dramatically elevated. Furthermore, RT-PCR analysis on the cytokine array of IL-2, IL-4, IL-12, TNF-α, and TGF-β in recipient splenocytes implied the Th1 to Th2 polarization. Therefore, it is deducible that alteration in the proportions of different T-lymphocyte subsets may be one of the main mechanisms by which grafted MSCs suppress the ongoing immune responses in vivo. The study here might provide some new clues for the design of therapeutic approaches for MSC transplantation.