Genistein, a kind of phytoestrogen abundant in soybeans, is beneficial for alleviating non-alcoholic fatty liver disease (NAFLD), but the specific mechanism was not clearly understood. This study was designed to determine the effect of genistein on NAFLD and explore the possible mechanism. 36 male Sprague-Dawley rats were divided into 4 groups: the control group, high fat-high sucrose diet (HFS) group, HFS with 4mg/kg body weight genistein, and HFS with 8mg/kg body weight genistein. 12 weeks later, serum and hepatic lipid profiles, liver histopathological examination were characterized. The protein levels of liver AMP-activated protein kinase (AMPK), phosphorylation of AMPK (p-AMPK), acetyl-CoA carboxylase (ACC), phosphorylation of ACC (p-ACC) and sterol regulatory element binding protein 1 (SREBP-1) were determined by western blot. mRNA expressions of fatty acid synthase gene (FAS) and glycerol-3-phosphate acyltransferase (GPAT), peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyl transfer enzyme-1 (CPT-1) and acyl-CoA oxidase (ACO) were measured by reverse transcription polymerase chain reaction (RT-PCR). Results showed that genistein effectively improved serum and hepatic lipid metabolism and diminished fat accumulation in liver. And the protein level of hepatic p-AMPK and p-ACC were increased, but SREBP-1 was decreased by genistein. Meanwhile, the mRNA levels of FAS and GPAT were lower, but PPARα, CPT-1, ACO were higher in rats treated with genistein compared with HFS group. Collectively, genistein can improve hepatic steatosis via activating AMPK, thus promoting fatty acid oxidation and inhibiting lipid synthesis in liver.