Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 mu g/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 mu g/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 mu g/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. (C) 2017 Published by Elsevier Inc.