In this study, the antioxidative effects of a purified polysaccharide isolated from the stems of Acanthopanax koreanum Nakai (ASP) on hydrogen peroxide-induced oxidative stress was investigated both in vitro and in vivo using a zebrafish model. A. koreanum Nakai stem was hydrolyzed using five carbohydrases and five proteases for the enzyme-assistant extraction. Of the enzyme-assistant extracts, the Protamex extract exhibited the highest yield and a potent scavenging activity against free radicals. Ethanol-added separation and anion exchange chromatography were conducted to identify the active polysaccharide. The purified polysaccharide significantly scavenged hydrogen peroxide and reduced hydrogen peroxide-induced cell death in Vero cells and in zebrafish. The results reveal that ASP is a useful antioxidant polysaccharide and might be available for relevant industrial applications.