Objective: Irisin is a novel exercise-induced myokine suggested to induce browning of white adipocytes. Myostatin involvels in the regulation of skeletal muscle mass and glucose metabolism. Recently, myostatin was negatively associated with irisin and its precursor FNDC5 expression in skeletal muscle. In our previous study, we observed that metformin inhibited myostatin gene expression in C2C12 myotubes. We determined if treatment with metformin modifies the expression and signaling pathway of the irisin in C2C12 cells. Methods: C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC; Manassas. VA) and grown in DMEM supplemented with 10% FBS. At confluence, myoblasts were induced to fuse into myotubes by using DMEM containing 2% horse serum (differentiation medium) for 4 days. After differentiation for 4 days, C2C12 myotubes were incubated with TGFß-1 (0, 2.0, and 5.0 ng/mL) for 24h in the serum-free medium. The irsin protein was determined by Western blot. Results: Metformin inhibits myostatin up-regulation induced by TGFß-1. Western blot analysis showed that metformin enhances the expression of irisin levels in C2C12 myotubes. Exogenous TGFß-1 significantly increased levels of phosphorylated Smad3 and decreased irisin expression in C2C12 myotubes. Pretreatment with metformin (2 mmol/L for 2 h) inhibited this process. Another AMPK activator, AICAR, did not alter myostatin and irisin expression induced by TGFß-1 in C2C12 myotubes. The AMPK inhibitor Compound C did not reverse the effect of metformin on myostatin and irisin expression. Conclusion: These results suggest that metformin enhanced irisin expression in C2C12 myotubes probably via inhibition of the TGFß-1 signalling pathway, independent of AMPK activation. These findings provide a possible explanation for the positive effect of metformin on muscle-fat crosstalk.