Myostatin is a transforming growth factor β superfamily member and is known as an inhibitor of skeletal muscle cell proliferation and differentiation. Exposure to myostatin induces G1 phase cell cycle arrest. In this study, we demonstrated that myostatin down-regulates Cdk4 activity via promotion of cyclin D1 degradation. Overexpression of cyclin D1 significantly blocked myostatin-induced proliferation inhibition. We further showed that phosphorylation at threonine 286 by GSK-3β was required for myostatin-stimulated cyclin D1 nuclear export and degradation. This process is dependent upon the activin receptor IIB and the phosphatidylinositol 3-kinase/Akt pathway but not Smad3. Insulin-like growth factor 1 (IGF-1) treatment or Akt activation attenuated the myostatin-stimulated cyclin D1 degradation as well as the associated cell proliferation repression. In contrast, attenuation of IGF-1 signaling caused C2C12 cells to undergo apoptosis in response to myostatin treatment. The observation that IGF-1 treatment increases myostatin expression through a phosphatidylinositol 3-kinase pathway suggests a possible feedback regulation between IGF-1 and myostatin. These findings uncover a novel role for myostatin in the regulation of cell growth and cell death in concert with IGF-1.