Abstract: Escherichia coli K5 strain was cultivated by fermentation, the supernatant was treated by ultrafiltration, exopolysaccharide (EPS) was extracted by ethanol precipitation, and then purified by Sevage method, dialysis, freeze-dried, DEAE-Sepharose and Sephadex G-75 column, finally high-purity K5 polysaccharide (K5) was obtained. N site of the K5 polysaccharide was chemically sulfated by Sulfur trioxide pyridine complex, NS-K5 polysaccharide (NS-K5) was prepared, and disaccharide analysis revealed that N-sulfation rate was up to 57%. The antioxidant capacity of K5 polysaccharideand N-sulfated K5 polysaccharide was investigated, the results showed that:within a certain concentration range, the reducing activity of NS-K5 was powerful over K5, when the concentration of K5 and NS-K5 was up to 1mg/mL, the hydroxyl radical scavenging rates were 17.7%, 25.6%, and DPPH radical scavenging rates were 26.1%, 45%, respectively. The result indicated that NS-K5 showed stronger antioxidant activity than K5.