In order to investigate the mechanisms by which puerarin from kudzu root extract regulates lipid metabolism, fifty mice were randomly assigned to five groups: normal diet, high-fat diet (HFD), and HFD containing 0.2%, 0.4% or 0.8% puerarin for 12 weeks. Body weight, intra-peritioneal adipose tissue (IPAT) weight, serum biochemical parameters, and hepatic and feces lipids were measured. Activity and mRNA and protein expressions of hepatic lipid metabolism-related enzymes were analyzed. Compared with HFD, 0.4% and 0.8% puerarin significantly decreased body and IPAT weight. There was a significant decrease in the serum and hepatic concentrations of total cholesterol, triglycerides and leptin in mice fed the 0.4% and 0.8% puerarin diets compared with HFD. Fatty acid synthase activity was suppressed in mice fed the 0.4% and 0.8% puerarin diets, while the activities of AMP-activated protein kinase (AMPK), carnitine acyltransferase (CAT) and hormone-sensitive lipase (HSL) were increased. mRNA expression of peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) was down-regulated in liver of mice fed the 0.8% diet compared with HFD, while mRNA expression of CAT and HSL was considerably up-regulated by 0.4% and 0.8% puerarin diets. The protein expression of PPAR gamma 2 in liver was decreased and those of p-AMPK, HSL and p-HSL were increased in mice fed 0.4% and 0.8% puerarin diets. These results suggest that > 0.4% puerarin influenced the activity, mRNA and protein levels of hepatic lipid metabolism-related enzymes, decreasing serum and liver lipids, body weight gain and fat accumulation. Puerarin might be beneficial to prevent lifestyle-related diseases.