Myostatin, a member of the transforming growth factor-␤ superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the ␥-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway. p21; MyoD; Notch; Hes1; Smad3 MYOSTATIN IS A CIRCULATING growth factor belonging to the transforming growth factor-␤ superfamily. Studies of myostatin function, conducted mostly in rodent (17-21, 29), bovine (14, 23), and chicken models (1, 2), have identified this protein as a negative regulator of myogenesis, but to date, few analyses have been directed to understanding the mechanism(s) underlying the role of myostatin in human skeletal muscle growth. Consistent with the inhibitory effect of myostatin in muscle growth, a mutation in the human myostatin gene results in muscular hypertrophy (25), and several studies have linked increased expression of myostatin with various human conditions that result in skeletal muscle wasting. For example, human immunodeficiency virus-infected men undergoing skeletal muscle wasting have increased intramuscular and serum levels of a myostatin-immunoreactive protein as compared with healthy controls (12), and human primary myoblasts from Duchenne muscular dystrophy patients show enhanced expression of myostatin (33); increased expression of serum myostatin-immunoreactive protein has also been correlated with advancing sarcopenia (32). In studies of the effect o...