Objective To investigate the effects of mitogen-activated protein kinase (MAPK) signaling pathway on B7H1-Ig fusion protein-initiated differentiation of type 1 regulatory T cells (Tr1). Methods Fresh isolated naive CD4+CD62L+T cells of C57BL/6 mice were stimulated with immobilized B7H1-Ig fusion protein plus anti-CD3 monoclonal antibody to induce Tr1 cells. The activities of three major MAPK (ERK1/2, p38MAPK, JNK) signaling pathways during the process of Tr1 cells differentiation were analyzed by Western blotting. In experiments, the specific inhibitors of ERK1/2, p38 and JNK (PD98059, SB203580 and SP600125) were added separately at the time of culture initiation, and the influences on cytokines secretion, immune function and Foxp3 expression of B7H1-Igstimulated CD4+T cells were detected respectively by ELISA, mixed lymphocyte reaction (MLR), flow cytometry and Western blotting. Results B7H1-Ig-plus-anti-CD3 stimulation activated and induced the generation of a subset of IL-10+IFN-γ+IL-5+IL-4low/-IL-2low/-Foxp3- Tr1 cells, which played immunosuppressive roles via secreting IL-10. Western blotting indicated that B7H1-Ig activated the p38 MAPK signaling pathway in CD4+T cells, while had no significant effect on ERK1/2 and JNK signaling pathways. Blocking p38 MAPK activity could significantly inhibit the production of IL-10 and IL-5 induced by B7H1-Ig in CD4+T cells, weaken the immunosuppressive function of B7H1-Ig-stimulated CD4+T cells and promote them to differentiate into CD25+Foxp3+Tregs. Conclusion The activation or inhibition of p38 MAPK signaling pathway is one of the important molecule mechanisms to control B7H1-Ig-induced Tr1 cells differentiation and transformation between Tr1 cells and CD4+CD25+Foxp3+Tregs.