Oxygen conditions influence a variety of biological processes of cells, including alterations in cellular growth, survival and differentiation. However, there have been few studies on the effects of hypoxia on proliferation and differentiation of myoblasts. In this study, we observed the effects of hypoxia (3% O(2)) on myogenic differentiation of C2C12 myoblasts and sought a possible mechanism involved in the regulation of myogenic differentiation of myoblasts by hypoxia. The expression of myosin heavy chain was detected by immunocytochemistry staining and Western blot analysis. The expression of muscle regulatory transcription factors was examined by RT-PCR analysis and Western blot analysis. The activity of extracellular signal-regulated kinases (ERK1/2) was investigated by forced expression of mitogen-activated protein kinase kinase 1 (MEK1(E)) and using Western blot analysis. We found that hypoxia inhibited myogenic differentiation of myoblasts. The expression of muscle regulatory transcription factors was downregulated by hypoxia in C2C12 myoblasts. During the inhibition of myogenic differentiation by hypoxia, ERK1/2 activation was suppressed, and increasing ERK1/2 activity by forced expression of MEK1(E) could partly reverse the inhibition of myogenic differentiation by hypoxia. These results suggest that the ERK1/2-mitogen-activated protein kinase pathway might be a possible mechanism involved in the inhibition of myogenic differentiation by hypoxia.