Objective：To investigate the role of P38 mitogen-activated protein kinases（P38MAPK） signal pathway in the differentiation of bone morphogenetic protein 9（BMP9） induced C3H10T1/2 stem cell into myocardiocytes. Methods：The phospho-P38MAPK and P38MAPK were measured by Western blot at 24 h after the C3H10T1/2 stem cell being transfected with pAdEasyBMP9 and the phos－pho-P38MAPK was detected by immunofluorescence at 24 h after the C3H10T1/2 stem cell being transfected with pAdEasyBMP9. The cardiac-specific proteins cTnT and CX43 were measured by Western blot，the cardiac-specific proteins cTnT and α-MHC were measured by immunofluorescence，the cardiac-specific gene GATA4 and MEF2C expressions were detected by RT-qPCR at 21 d after the C3H10T1/2 stem cell being transfected with pAdEasyBMP9 and treated with SB203580. Results：Transfection with pAdEasyBMP9 produced rapid increase in phospho-P38MAPK and little change in P38MAPK. Phospho-P38MAPK expressed in nucleus and cytoplasm or in different states of nucleus. SB203580 significantly inhibited the pAdEasyBMP9-induced expression of CX43，cTnT，α-MHC，GATA4，MEF2C in C3H10T1/2 stem cell. Conclusions：P38MAPK signal pathway can be activated in C3H10T1/2 stem cell after being transfected with pAdEasyBMP9 and can promote the myocardiocytes differentiation.