Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. Myostatin, a negative regulator of myogenesis, inhibits myoblast differentiation by suppressing the expression of MyoD, Myf5, myogenin, and p21. This inhibition is mediated through Smad3, which interacts with MyoD and reduces its activity. Myostatin treatment leads to the down-regulation of MyoD and other myogenic regulators, preventing myoblast differentiation into myotubes. The effect of myostatin on myoblast differentiation is reversible, as myoblasts can differentiate again once myostatin is removed. Myostatin-treated myoblasts are functionally different from reserve cells, which remain quiescent and undifferentiated but retain the ability to proliferate and differentiate. Myostatin also inhibits p21 expression, which is involved in cell cycle regulation. Despite the forced expression of MyoD, myostatin still inhibits myogenic differentiation, indicating that MyoD is not sufficient to rescue the inhibition. Myostatin activates Smad3, which interacts with MyoD and reduces its activity, leading to the inhibition of myogenic differentiation. These findings suggest that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in myostatin-null mice and myostatin-mutant cattle are due to deregulated proliferation and differentiation of myoblasts.Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. Myostatin, a negative regulator of myogenesis, inhibits myoblast differentiation by suppressing the expression of MyoD, Myf5, myogenin, and p21. This inhibition is mediated through Smad3, which interacts with MyoD and reduces its activity. Myostatin treatment leads to the down-regulation of MyoD and other myogenic regulators, preventing myoblast differentiation into myotubes. The effect of myostatin on myoblast differentiation is reversible, as myoblasts can differentiate again once myostatin is removed. Myostatin-treated myoblasts are functionally different from reserve cells, which remain quiescent and undifferentiated but retain the ability to proliferate and differentiate. Myostatin also inhibits p21 expression, which is involved in cell cycle regulation. Despite the forced expression of MyoD, myostatin still inhibits myogenic differentiation, indicating that MyoD is not sufficient to rescue the inhibition. Myostatin activates Smad3, which interacts with MyoD and reduces its activity, leading to the inhibition of myogenic differentiation. These findings suggest that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in myostatin-null mice and myostatin-mutant cattle are due to deregulated proliferation and differentiation of myoblasts.