AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMPK activating compounds that have a much lower effective concentration than metformin. As a result, Emodin, a natural anthraquinone derivative was shown to stimulate AMPK activity in skeletal muscle and liver cell. Emodin enhanced GLUT4 translocation and [14C] glucose uptake into myotube in an AMPK dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species (ROS) and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activity. Finally, we confirmed that a single-dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for eight days using an insulin tolerance test and insulin stimulated PI-3K phosphorylation in wild type and HFD induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of Type 2 diabetic models.