To increase the production of Grifola frondosa polysaccharides (GFPs), a hydrogen peroxide/ascorbic acid (H2O2/Vc)-assisted extraction method was established. The extraction conditions were optimized by response surface methodology. Purification of the crude GFP by DEAE-52 and Sephadex G100 column chromatography yielded three fractions, GFP0, GFP1, and GFP3. Monosaccharide composition analysis revealed that these GFPs mainly consisted of mannose, glucose, galactose and l-fucose, together with a low proportion of xylose, glucuronic acid and ribose. Chemical antioxidant assays indicated that these GFPs possessed considerable reducing power and radical (DPPH<middle dot>, HO<middle dot> and ABTS<middle dot>+) scavenging abil-ity. They were also observed to availably protect RAW264.7 cells from H2O2-induced oxidative stress, reduce intracellular ROS and MDA levels, and increase intracellular SOD and CAT activities. These GFPs were able to up-regulate the mRNA expression of SOD1, CAT, HO-1 and Nrf2, indicating that regulation of antioxidant enzymes and Nrf2 signaling pathway could be their important antioxidant mechanism.