Abstract: Objective:Four different extraction methods(alkali extraction,acid extraction,enzymatic extraction,and hot water extraction)were used to extract polysaccharides from superfined wheat bran under the same extract conditions. The extraction yield,uronic acid contents,sulfate contents,protein contents,monosaccharide composition,and the immunoregulatory activities of four samples were investigated. Methods:The total carbohydrate and protein contents were determined by the phenol-sulfuric acid method and the BCA kit. Uronic acid content of the polysaccharide was determined by sulfamate/m-hydroxydiphenyl assay. The sulfate content of the polysaccharide was determined by the BaCl₂ gelatin method and quantitative determination of the monosaccharide composition of the polysaccharides was performed using gas chromatography system. Cell viability was evaluated by MTT assay and nitric oxide(NO)content was determined by Griess reagent assay. Moreover,the expression of iNOS and COX-2 was determined by western blot. Results:Comparing different extraction methods,polysaccharide by alkali extraction had the highest extraction yield,which reached up to 31.73%;total polysaccharides content was 57.79%. The contents of uronic acid and sulfate of four different samples were ranked as followed order:alkali extraction,enzymatic extraction,acid extraction,and hot water extraction. Monosaccharide composition of wheat bran polysaccharides were analyzed by GC-MS and rhamnose,arabinose,and glucose by GC system under different extraction. Four different samples didn’t show any cytotoxicity on RAW 264.7 cells and the polysaccharide prepared by water-extraction could significantly promote RAW264.7 cells to produce NO production. Furthermore,it could increase iNOS and COX-2 protein expression. Conclusion:The chemical composition and biological activity of wheat bran polysaccharide by different extraction methods were different,it was more beneficial to maintain the functional activity of polysaccharides based on the traditional water-extraction technology.