The study was purposed to investigate the effects and mechanism of bone marrow-derived mesenchymal stem cells (MSCs) on graft-versus-host desease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The model of GVHD in rat had been established by allo-HSCT with donor derived T cells. The occurence of GVHD in recipients was observed in condition with or without donor derived MSC co-transplantation. Effects of MSCs on GVHD were analyzed by model rat survival rate and pathology. Proportions of CD4+CD25+ regulatory T cells were determined by using label spleen lymphocytes and thymocytes with double fluorescent-labeled antibodies and flow cytometry. The results showed that MSCs inhibited the lethal GVHD after HSC co-transplantation and increased the survival rate. The ratio of CD4/CD8 deceased in GVHD group in different levels, as compared with that in the experimental group. The proportion of CD4+CD25+ regulatory T cells of spleen lymphocytes was 31.55 +/- 7.58% and 20.90 +/- 1.90% in experimental and GVHD groups, respectively. Similarly, the proportion of CD4+CD25+ regulatory T cells of thymocytes was 93.20 +/- 2.69% and 57.17 +/- 6.79% in experimental and the GVHD groups, respectively. Meanwhile the proportion of CD4+CD25+ regulatory T cells was higher in experimental group than that in GVHD group. It is concluded that MSCs may prevent the lethal GVHD after allo-HSC co-transplantation and raise the survival rate of model rats by acting on the CD4+CD25+ regulatory T cells in vivo.