The present study was to characterize the avenanthramide-rich extract (ARE) from oat bran and assess its effect on activity and gene expression of antioxidant enzymes in D-galactose-induced oxidative-stressed mice. High-performance liquid chromatography (HPLC) analysis found that ARE had 6.07% N-(3',4'-dihydroxycinnamoyl)-5-hydroxyanthranilic acid (Bc), 4.37% N-(4'-hydroxycinnamoyl)-5-hydroxyanthranilic acid (Bp), and 5.36% N-(4'-hydroxy-3'-methoxycinnamoyl)-5-hydroxyanthranilic acid (Bf). In addition, ARE was also rich in vanillic acid (0.60%), caffeic acid (0.50%), syringic acid (0.54%), p-coumaric acid (0.16%), ferulic acid (0.08%), and sinapic acid (0.03%). Administration of D-galactose markedly lowered not only the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) but also the gene expression of manganese superoxide dismutase (SOD), copper-zinc SOD, glutathione peroxidase (GPx), and lipoprotein lipase (LPL) mRNA in mice. Administration of ARE significantly reversed the D-galactose-induced oxidative stress by increasing the activity of the antioxidant enzymes and upregulating their gene expression. This was accompanied by a significant decrease in the malondialdehyde (MDA) level in mice given ARE compared to the control. The results demonstrated that ARE possessed the antioxidant activity and was effective against D-galactose-induced oxidative stress.