We analyzed the role of Hypoxia‐inducible factor (HIF)‐1α in myoblast differentiation by examining the expression and regulation of HIF‐1α in proliferating and differentiating C2C12 myoblast, and by knocking down HIF‐1α of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF‐1α has been shown to be involved in differentiative process in non‐muscle tissues. Although HIF‐1α mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF‐1α protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF‐1α accumulated in the nuclei of myogenin‐positive myoblasts. The inhibition of proteasome in the growth phase led to HIF‐1α protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF‐1α, suppressed HIF‐1α accumulation. Therefore, we suggest that the level of HIF‐1α protein expression is regulated by a proteasome‐and chaperon‐dependent pathway in C2C12 myoblast. Knockdown of HIF‐1α effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF‐1α expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF‐1α is required for C2C12 myogenesis in vitro, and suggest that HIF‐1α may have an essential role in regenerative muscle tissue in vivo. J. Cell. Biochem. 98: 642–649, 2006. © 2006 Wiley‐Liss, Inc.