Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor beta (TGF-beta) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-beta family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration ofmono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.