Ulmus macrocarpa Hance as an oriental medicinal plant has shown enormous potential for the treatment of several metabolic disorders in Korea. Hyperlipidemia, which is characterized by the excess accumulation of lipid contents in the bloodstream, may lead to several cardiovascular diseases. Therefore, in this study, anti-hyperlipidemic potential of U. macrocarpa water extract (UME) was examined in vitro and in vivo using HepG2 cells and experimental rats, respectively. The hyperlipidemia in experimental rats was induced by the high-cholesterol diet (HCD) followed by oral administration of various concentrations (25, 50 and 100 mg/kg) of UME for 6 weeks. As a result, the UME significantly improved the biochemical parameters such as increased the level of triglyceride, total cholesterol, and low-density lipoprotein cholesterol as well as reduced the high-density lipoprotein cholesterol in the HCD-fed rats. In addition, UME also prevented lipid accumulation through regulating AMPK activity and lipid metabolism proteins (ACC, SREBP1 and HMGCR) in the HCD-fed rats as compared to the controls. Moreover, similar pattern of gene expression levels was confirmed in oleic acid (OA)-treated HepG2 cells. Taken together, our results indicate that UME prevents hyperlipidemia via activating the AMPK pathway and regulates lipid metabolism. Thus, based on the above findings, it is estimated that UME could be a potential therapeutic agent for preventing the hyperlipidemia.