Radix Pueraria lobata (RP) has been reported to prevent obesity and improve glucose metabolism; however, the mechanism responsible for these effects has not been elucidated. The mechanism underlying anti-obesity effect of RP was investigated in high-fat diet (HFD) induced obese mice and skeletal muscle cells (C2C12). Five-week-old C5BL/6 mice were fed a HFD containing or not containing RP (100 or 300 mg/kg) or metformin (250 mg/kg) for 16 weeks. RP reduced body weight gain, lipid accumulation in liver, and adipocyte and blood lipid levels. In addition, RP dose-dependently improved hyperglycemia, insulinemia, and glucose tolerance, and prevented the skeletal muscle atrophy induced by HFD. Furthermore, RP increased the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle tissues. RP and its main component, puerarin, increased mitochondrial biogenesis and myotube hypertrophy in C2C12 cells. The present study demonstrates that RP can prevent diet-induced obesity, glucose tolerance, and skeletal muscle atrophy in mouse models of obesity. The mechanism responsible for the effect of RP appears to be related to the upregulation of energy metabolism in skeletal muscle, which at the molecular level may be associated with PGC-1α and AMPK activation.