OBJECTIVE To investigate the effect of cyclic stretch on p38 mitogen-activated protein kinase (p38MAPK) signaling pathway during stretch-induced differentiation process of C2C12 myoblasts. METHODS C2C12 cells were seeded on Bio Flex 6-well plates, and cells were subsequently subjected to cyclic stretch at an optimal magnitude (10%) and frequency (0.5 Hz). The effects of cyclic stretch were examined at 2, 6, 12, 24 h. Antibodies specific to p38MAPK phosphorylated forms and the total protein levels of the p38MAPK were examined using Western blot analysis. RESULTS These results indicated that p38MAPK was activated during stretch-induced C2C12 cell differentiation. The level of phosphorylated protein was higher in the p38MAPK signaling pathway. The expression of total protein was maintained at baseline level. There were no significant differences between groups. Treatment of cells with specific p38MAPK inhibitor SB203580 could decrease the expression of myogenin, but not completely abolish the myogenin expression after stretch. CONCLUSION p38MAPK signaling pathway plays an important role during stretch-induced differentiation process of C2C12 myoblasts, but is not activated exclusively in this process.