Objective：To investigate the role of ERK5 signal pathway in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9（BMP9）. Methods：C3H10T1/2 cells were infected with recombinant adenovirus AdBMP9 and AdGFP，and the transfection efficiency was detected by flow cytometry. Expression levels of phosphorylated ERK5（p-ERK5） and ERK5 were detected by Western blot and the expression sites of p-ERK5 in the cell were detected by immunofluores－cence. C3H10T1/2 cells were preprocessed with ERK5 special inhibitor BIX02189 and induced by AdBMP9. Expression levels of p-ERK5 and ERK5 were detected by Western blot；expression levels of myocardial specificity gene myocyte enhancer factor 2C（MEF2C） and GATA4 were measured by real-time fluorescence quantitative PCR and expression levels of myocardial specificity protein connexin 43（CX43） and cardiac troponin T（cTnT） were detected by Western blot. Results：The transfection efficiencies of AdGFP and AdBMP9 were about 50% and the expression level of p-ERK5 after being induced by AdBMP9 was significantly higher（P<0.01）. Immunofluorescence showed that the expression of p-ERK5 of BMP9 group was obviously increased and mainly concen－trated in the nucleus. 15 ?滋mol/L BIX02189 completely inhibited the expression of p-ERK5（P<0.01） and significantly inhibited the AdBMP9 induced expression of MEF2C，GATA4 genes and CX43，cTnT proteins in C3H10T1/2 cells（P=0.000）. Conclusion：The ERK5 signal pathway can be activated by BMP9 to promote cardiomyocyte-like differentiation from C3H10T1/2 cells.