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Fat accumulation in the liver increases the risk of developing progressive liver injury. It can induce all the symptoms of metabolic syndrome, which is associated with many additional health problems, including increased risk of obesity, hypertension, insulin resistance, and non-alcoholic fatty liver disease (NAFLD). Therefore, prevention and treatment of fat accumulation in the liver are relevant to health promotion. Mulberry leaf polyphenol extracts (MLPE) have been known to modulate serum fasting glucose, lipid and antiatherosclerosis. However, the effect of MLPE on regulating hepatic lipid metabolism is unclear. This study evaluated the effects and mechanisms of MLPE in reducing hepatic lipid accumulation in cell culture. We found MLPE could regulate hepatic lipid accumulation. Further, numerous lipogenic enzymes, such as FAS (fatty acid synthetase), ACC (acetyl-CoA carboxylase), HMGCR (HMG-CoA reductase) and associated-lipogenic transcriptional factors (SREBP1 and SREBP2) were suppressed by MLPE. Our results show MLPE is able to reduce hepatic lipid accumulation through activation of the AMPK (AMP-activating protein kinase) signaling pathway. It may have potential therapeutic implications for human NFALD.