Abstract: Excessive lipid accumulation within liver has been proposed to cause obesity, hyperlipidemia, diabetes, and fatty liver disease. Rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, has various biological functions, including antioxidant, anti-inflammatory, and anticancer effects. However, a hypolipidemic effect of rutin on fatty liver disease has not been reported. In this study, we examined the effect of rutin on reducing lipid accumulation in hepatic cells. Hepatocytes were treated with oleic acid (OA) containing with or without rutin to observe the lipid accumulation by Nile red stain. The result showed rutin suppressed OA-induced lipid accumulation and increased adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) activity in hepatocytes. The expression of critical molecule involved in lipid synthesis, sterol regulatory element binding proteins-1 (SREBP-1), was attenuated in rutin-treated cells. Moreover, long-term incubation of rutin inhibited the transcriptions of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), glycerol-3-phosphate acyltransferase (GPAT), fatty acid synthase (FAS), and acetyl-coenzyme carboxylase (ACC). Besides, we also found out the antioxidative effect of rutin by increasing the expression of peroxisome proliferator-activated receptor (PPAR)-α and antioxidative enzymes. Taken together, our findings suggest rutin could attenuate lipid accumulation by decreasing lipogenesis and oxidative stress in hepatocyte.