Obesity-induced hepatic lipid accumulation is a crucial factor in the initiation and development of nonalcoholic fatty liver disease. In this study, we investigated the effect of isorhamnetin, a naturally occurring flavonoid in fruits and vegetables, on free fatty acid (FFA)-induced lipid accumulation in hepatocytes. Hepatocytes (Hepa1c1c) were exposed to FFA with or without isorhamnetin, and lipid accumulation was estimated by Oil Red O staining and biochemical measurement. The metabolic genes/proteins involved in lipid metabolism were measured by qRT-PCR and/or western blotting. Isorhamnetin significantly reduced lipid accumulation in FFA-induced hepatocytes, and this was accompanied by modulation of genes involved in lipid metabolism. Isorhamnetin up-regulated expression of fatty acid oxidation-related genes (peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1, and acyl-CoA oxidase), and down-regulated lipogenic gene expression (sterol regulatory element-binding protein-1, fatty acid synthase, and acetyl-CoA carboxylase). Alterations in the metabolic gene expression induced by isorhamnetin were blunted by PPARα antagonist. These findings indicate that the inhibitory action of isorhamnetin in FFA-induced lipid accumulation in hepatocytes is associated with the PPARα pathway. Isorhamnetin may be useful for protection against obesity-related hepatosteatosis.