Myostatin, a member of transforming growth factor family, has been identified as a master regulator of embryonic myogenesis and early post-natal skeletal muscle growth. However, cumulative evidences also suggest that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression and that myostatin may contribute to muscle mass loss in adulthood. Two major branches of the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammalian target of rapamycin (mTOR) pathway, which controls protein synthesis, and the Akt/FOXO pathway, which controls protein degradation. Here, we provide further insights into the mechanisms by which myostatin regulates skeletal muscle mass by showing that myostatin negatively regulate Akt/mTOR signaling pathway. Electrotransfer of a myostatin expression vector into the Tibialis anterior muscle of Sprague Dawley male rats increased myostatin protein level and decreased skeletal muscle mass 7 d after gene electrotransfer. Using RT-PCR and immunoblots analyses, we showed that myostatin overexpression was ineffective to alter the ubiquitin-proteasome pathway. By contrast, myostatin acted as a negative regulator of Akt/mTOR pathway. This was supported by data showing that the phosphorylation of Akt on Thr308, tuberous sclerosis complex 2 on Thr1462, ribosomal protein S6 on Ser235/236 and 4E-BP1 on Thr37/46 was attenuated 7 d after myostatin gene electrotransfer. The data support the conclusion that Akt/mTOR signaling is a key target that accounts for myostatin function during muscle atrophy, uncovering a novel role for myostatin in protein metabolism and more specifically in the regulation of translation in skeletal muscle.