Objective: To investigate the effects of metformin treatment on fatty acid metabolism and expression and activity of AMP-activated protein kinase (AMPK)α in skeletal muscle in type 2 diabetes. Methods: A total of 30 cases with or without type 2 diabetes mellitus (T2DM) who received selective surgery were enrolled from department of orthopaedics. These patients were divided into three groups: DM group, DM+ MET group and NC group. Blood samples were drawn from an antecubial vein for measurement of plasma glucose, insulin, lipid and free fatty acid (FFA). Insulin sensitivity index (ISI) was calculated. Skeletal muscle was freezed in liquid nitrogen during orthopaedics surgery and was kept at -70 ℃ until assay. Skeletal muscle tissue was analysed en bloc for triglyceride, long chain fatty acyl-CoA (LCACoAs), AMPKα1 and AMPKα2 mRNA expression [quantitative real-time polymerase chain reaction (qRT-PCR)], as well as protein expression of AMPKα1, AMPKα2 and phosphorylated AMPKα (p-AMPKα) (Western blot). Results: ⑴ Compared with NC group, HbA1c, fasting blood glucose (FBG), fasting insulin (FINS), FFA and triglyceride(TG) were increased in DM group, while metformin treatment decreased FFA and TG; ISI was reduced in DM group than that in NC group, but ISI was higher in DM+ MET group compared with DM group (P<0.05 or P<0.01). ⑵ The contents of triglyceride and LCACoAs in skeletal muscle in DM group were higher than those in NC group, while those in DM + MET group were lower than those in DM group (P<0.05 ). ⑶ No alternations of mRNA levels and protein levels of AMPKα1 were detected among three groups (P>0.05). Compared with NC group, AMPKα2 mRNA expression and protein levels of AMPKα2 and p-AMPKα in skeletal muscle were decreased in DM group, while metformin treatment increased protein level of p-AMPKα (P<0.05). Conclusions Compared with NC group, muscle lipid accumulation and insulin resistance always exist in T2DM patients. Metformin treatment may increase activity of AMPKα, resulting in a decrease in lipid accumulation in skeletal muscle and increase insulin sensitivity in T2DM patients.