Abstract: AIM To investigate the effect of irisin on glucose uptake in C2C12 myocytes and its underlying mechanism. METHODS C2C12 myocytes were divided randomly into the following groups: Control, irisin (0.1 μg/ml, 0.3 μg/ml, 1 μg/m, respectively), HF/HG, HF/HG+irisin (0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, respectively), Control+scramble siRNA, HF/HG+AMPKα2 siRNA, HF/HG+scramble siRNA, HF/HG+scramble siRNA+irisin, and HF/HG+AMPKα2 siRNA+irisin. The phosphorylated and total AMPK and GLUT4 were determined by Western blot and the uptake of glucose was assayed by analyzing the fluorescence intensity of 2-NBDG. RESULTS irisin significantly increased glucose uptake in C2C12 myocytes in a concentration-dependent fashion both in HF/HG treated group and non-HF/HG treated group (all P<0.05). Compared with those in control group, HF/HG decreased the GLUT4 expression and irisin significantly increased GLUT4 expression (P<0.05). Compared with those in control group, HF/HG decreased the phosphorylated AMPK in C2C12 myocytes and irisin increased the phosphorylated AMPK both in HF/HG treated group and non-HF/HG treated group (P<0.05 or P<0.01). AMPKα2siRNA inhibited the increase of glucose uptake and phosphorylation of AMPK induced by irisin in C2C12 myocytes (P<0.05 or P<0.01). CONCLUSION Irisin increases glucose uptake in myocytes and skeletal muscle by activating AMPK.