The aim of this study was to investigate the potential protective effects of the hot water extract of Eriobotrya japonica (EJW) on EtOH-or free fatty acid (FFA)-induced fatty liver injury in vitro. HepG2/2E1 cells were exposed to EtOH and HepG2 cells were exposed to a mixture of FFAs (oleic acid:palmitic acid, 2:1) to stimulate oxidative stress and to induce lipid accumulation, respectively. Antioxidant activity was significantly increased and lipid accumulation was inhibited in cells pretreated with EJW compared to those in cells exposed to EtOH or FFA only. Also, 5 0 adenosine monophosphate (AMP)activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylations were considerably increased, indicating activation of AMPK. Furthermore, EJW reduced the messenger RNA (mRNA) expression of lipogenesis-associated factors such as ACC, sterol regulatory element binding protein-1c (SREBP-1c), and fatty acid synthase (FAS), and increased mRNA expression related to components of the fatty acid b-oxidation pathway, such as AMPK, carnitine palmitoyltransferase 1 (CPT-1), and peroxisome proliferator-activated receptor alpha (PPARa). These results suggest that EJW possessed potential preventive effects against both EtOH-and FFA-induced fatty liver disease by alleviation of oxidative stress and lipid accumulation in hepatocytes.