The effect and potential molecular mechanisms of berberine on gluconeogenesis in skeletal muscles and adipose tissues were investigated. After adaptive feeding for one week, 8 rats were randomly selected as the normal group and fed on a standard diet. The remaining 32 rats were fed on a high-fat diet and given an intravenous injection of streptozotocin (STZ) for 2 weeks to induce the diabetic models. The diabetic rat models were confirmed by oral glucose tolerance test (OGTT) and randomly divided into 4 groups (n=8 each), which were all fed on a high-fat diet. Berberine (3 g/kg per day) or metformin (183 mg/kg per day) was intragastrically administered to the diabetic rats for 12 weeks, serving as berberine group and metformin group respectively. 5-aminoimidazole-4-carboxamide1-beta-D-ribofuranoside [AICAR, an agonist of AMP-activated protein kinase (AMPK), 0.5 mg/kg per day] was subcutaneously injected to the diabetic rats for 12 weeks, serving as AICAR group. The remaining 8 diabetic rats served as the model group, which was given a 0.5% carboxyl methylcellulose solution by oral gavage. Fasting serum insulin (FINS), OGTT as well as lipid parameters were tested by commercial kit. The protein levels of liver kinase B1 (LKB1), AMPK, phosphorylated AMP-activated protein kinase (p-AMPK), transducer of regulated CREB activity 2 (TORC2), phosphorylated transducer of regulated CREB activity 2 (p-TORC2), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in skeletal muscles and adipose tissues were examined by Western blotting. The results showed that berberine significantly decreased the body weight, plasma glucose, insulin levels, and homeostatic model assessment for insulin resistance (HOMA-IR) of diabetic rats compared with those in the model group. Meanwhile, the serum total triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels were markedly decreased and high-density lipoprotein cholesterol (HDL-C) level was significantly increased after the treatment with berberine. In addition, we found that berberine significantly increased the expression of p-AMPK and LKB1, while decreasing the p-TORC2 levels in skeletal muscles and adipose tissues. Moreover, the expression of PEPCK and G6Pase was significantly down-regulated after the treatment with berberine compared to the model group. It was suggested that the mechanism by which berberine inhibited peripheral tissue gluconeogenesis may be attributed to the activation of the LKB1-AMPK-TORC2 signaling pathway.