Extracellular matrix (ECM) and its regulatory proteins such as matrix metalloproteinases (MMPs) in skeletal muscles are inextricably linked with regulation of muscle mass and function. ECMs has also been found to be involved in muscle diseases such as muscular dystrophy and muscle mass loss like sarcopenia. We found that batimastat, a broad spectrum MMP inhibitor promotes myosin heavy chain (MHC) expression during myogenic differentiation of C2C12 myoblasts. The expression of myogenin, which is a transcription factor that induces MHC expression, was also enhanced by batimastat. Batimastat promoted MHC expression even under conditions where myogenic differentiation was strongly inhibited, such as differentiation into other cell type (osteoblast) or proliferative cultures. Increased MHC expression was found in other MMP inhibitor possessing similar spectrum to batimastat, but not in specific inhibitors for MMP-3, -9 or -13 in C2C12 myoblasts, suggesting that several MMPs coordinately regulate MHC expression. Interestingly, batimastat-induced enhancement of MHC expression was attenuated in myogenic differentiation of senescent C2C12 myoblasts with elevated senescence-associated β-galactosidase activity. These results indicate that the mechanism regulating myoblast differentiation by MMPs may be altered by cellular senescence. Elucidating the senescence-induced changes in MMP-mediated regulation can contribute to the understanding of the pathogenesis of age-related muscle diseases such as sarcopenia.