This study was performed to investigate the action of pilose antler peptide (PAP) on high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) in rats. Animals were randomly divided into five groups: control group, model group receiving high-fat diet (HFD), atorvastatin group receiving high-fat diet (HFD) + atorvastatin (20 mg/kg), PAP-L group receiving HFD + PAP (50 mg/kg) and PAP-H group receiving HFD + DB (100 mg/kg). PAP administration significantly reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), with improvement in liver histopathological examination. To further investigate the molecular mechanism of the effect of PAP on NAFLD, expression levels of AMP-activated protein kinase (AMPK)/NF-κB pathway in the liver were determined by western blot analysis, respectively. These results suggested that PAP administration was beneficial to treatments of NAFLD in rats fed HFD by modulating the expression levels of AMPK/NF-κB pathway.