Myostatin, a member of the TGF-β superfamily, functions as a negative regulator of muscle growth by inhibiting myoblast proliferation. This study demonstrates that myostatin controls the progression of myoblasts through the cell cycle. When C2C12 myoblasts were exposed to increasing concentrations of myostatin, their proliferation decreased. Flow cytometry and Western blot analysis revealed that myostatin prevents myoblasts from progressing from the G1 to S phase of the cell cycle. Myostatin specifically up-regulates p21 (Waf1/Cip1), a cyclin-dependent kinase inhibitor (CKI), and decreases the levels and activity of Cdk2 protein in myoblasts. These changes lead to the accumulation of hypophosphorylated Rb protein, resulting in G1-phase arrest of myoblasts. This mechanism explains the generalized muscular hyperplasia observed in animals lacking functional myostatin. Myostatin inhibits myoblast proliferation by regulating the cell cycle, and its effects can be reversed by removing myostatin from the culture. Myostatin also affects the G2/M phase of the cell cycle. The study shows that myostatin up-regulates p21 expression and down-regulates Cdk2 activity, leading to the hypophosphorylation of Rb and G1-phase arrest of myoblasts. These findings suggest that the hyperplasia in myostatin-null mice and double-muscled cattle is due to deregulated myoblast proliferation. The results highlight the role of myostatin in controlling muscle growth through the regulation of myoblast proliferation and cell cycle progression.Myostatin, a member of the TGF-β superfamily, functions as a negative regulator of muscle growth by inhibiting myoblast proliferation. This study demonstrates that myostatin controls the progression of myoblasts through the cell cycle. When C2C12 myoblasts were exposed to increasing concentrations of myostatin, their proliferation decreased. Flow cytometry and Western blot analysis revealed that myostatin prevents myoblasts from progressing from the G1 to S phase of the cell cycle. Myostatin specifically up-regulates p21 (Waf1/Cip1), a cyclin-dependent kinase inhibitor (CKI), and decreases the levels and activity of Cdk2 protein in myoblasts. These changes lead to the accumulation of hypophosphorylated Rb protein, resulting in G1-phase arrest of myoblasts. This mechanism explains the generalized muscular hyperplasia observed in animals lacking functional myostatin. Myostatin inhibits myoblast proliferation by regulating the cell cycle, and its effects can be reversed by removing myostatin from the culture. Myostatin also affects the G2/M phase of the cell cycle. The study shows that myostatin up-regulates p21 expression and down-regulates Cdk2 activity, leading to the hypophosphorylation of Rb and G1-phase arrest of myoblasts. These findings suggest that the hyperplasia in myostatin-null mice and double-muscled cattle is due to deregulated myoblast proliferation. The results highlight the role of myostatin in controlling muscle growth through the regulation of myoblast proliferation and cell cycle progression.