The objective of this study was to determine the antioxidant capacity of polyphenols extracted from black highland barley (BHLPE) in vitro and in vivo. BHLPE was found to have strong superoxide radical, hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, ferric reducing antioxidant power and moderate metal ion-chelating activity. Compared with a high fat diet (HFD) group, mice that were administered 600mg BHLPE/kg body weight showed significant decreases in total cholesterol, low-density lipoprotein cholesterol and the atherosclerosis index, in addition to markedly increased high-density lipoprotein cholesterol levels. Furthermore, the antioxidant defense system and antioxidant gene expression were significantly improved in vivo in mice that were administered BHLPE compared with mice in the HFD group. These results suggest that BHLPE has significant potential as a natural antioxidant to promote health and to reduce the risk of disease.