Follistatin (FS) inhibits several members of the TGF- superfamily, including myostatin (Mstn), a negative regulator of muscle growth. Mstn inhibition by FS represents a potential therapeutic approach against muscle atrophy. The goal of our study was to investigate the mechanisms of the FS-induced muscle hypertrophy. To test the role of satellite cells in the FS effect, we used irradiation to destroy their proliferation capacity. FS overexpression increased the muscle weight by about 37% in control animals, while the increase reached only 20% in irradiated muscle, supporting the role of cell proliferation in the FS-induced hypertrophy. Surprisingly, the muscle hypertrophy caused by FS reached the same magnitude in Mstn KO and in WT mice, suggesting that Mstn might not be the only ligand of FS involved in the regulation of muscle mass. To assess the role of activin (Act), another FS ligand, in the FS-induced hypertrophy, we electroporated FSI-I, a FS mutant which does not bind Act with high affinity. While FS electroporation increased muscle weight by 32%, the muscle weight gain induced by FSI-I reached only 14%. Furthermore, in Mstn KO mice, FSI-I overexpression failed to induce hypertrophy, in contrast to FS. These results suggest therefore that Act inhibition may contribute to FS-induced hypertrophy. Finally, the role of Act as a regulator of muscle mass was supported by the observation that ActA overexpression induced muscle weight loss (-15%). In conclusion, our results show that satellite cells proliferation and both Mstn and Act inhibition are involved in the FS-induced muscle hypertrophy.