Objective：To explore the effects of splenocytes on graft-versus-host disease（GVHD） and chimerism in murine mixed bone marrow transplantation. Methods：During mixed transplantation, syngeneic bone marrow cells from BALB／c mice were mixed with H-2 haploidentical matched bone marrow cells from CB6F1 mice. At the same time, different infusion dose of splenocytes were transplanted into lethally irradiated groups of BALB／c mice. BALB／c mice of group A hadn’t accepted splenocytes of CB6F1 mice. BALB／c mice of group B were infused with the same dose of syngeneic and CB6F1 mice splenocytes. BALB／c mice of group C accepted the double dose of splenocytes of CB6F1 mice compared with syngeneic splenocytes. After transplantation, the appearance of a GVHD, the level of the chimerism of the recipient mice were observed and detected by histopathological examination on liver, skin, small intestine and flow cytometry. Hematopoietic recovery and the mortality were evaluated by blood routine test and clinical observation. Results：Mice in mixed transplantation groups survived over 50 days. No GVHD manifestation and histopathological alterations appeared in group A, and the chimerism on the 150th day decreased significantly. Mice of group B showed little GVHD manifestation and histopathological changes, and the chimerism on the 150th day was reduced. Mice of group C showed a higher level and more stable of chimerism with grade Ⅱ degree of GVHD histopathological alterations. Typical GVHD manifestation and histopathological alterations appeared in H-2 haploidentical bone marrow transplantation group. The survival rate was only 20% 50 days after transplantation. Conclusion：The proportion and dose of syngeneic and haploidentical MHC splenocytes were of importance to balance GVHD and host-versus-graft reaction（HVGR）.