Nonalcoholic fatty liver disease (NAFLD) is a kind of liver inflammation caused by an accumulation of fat in the liver. Patients with NAFLD have an increased risk to develop liver fibrosis, which leads to cirrhosis. To investigate hepatoprotective effects of Agrimonia eupatoria L (A. eupatoria), oleic acid-induced NAFLD in HepG2 cells was used and A. eupatoria was fractionated with ethanol (EtOH), n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH), and H2O. Cells treated with the EtOAc fraction showed the highest lipid accumulation inhibiting effect. A. eupatoria also suppressed triglyceride accumulation and inhibited expression of lipid marker gene, such as a peroxisome proliferator activated receptor γ (PPAR-γ). Moreover, another marker, mRNA expression level of peroxisome proliferator activated receptor α (PPAR-α) was significantly increased by in a dose-dependent manner. These results suggest that A. eupatoria is a potent agent for the treatment of NAFLD.