The aim of the present study was to explore the immunomodulatory effects of sulfated K5 polysaccharide derivatives on RAW264.7 macro-phage cells， and to further elucidate the structure?activity relationship. In the present study， chemically sulfated polysaccharides were derived from Escherichia coli K5 capsular polysaccharide (K5PS)， and molecular weight determination， sugar analysis， and other physical and chemical characterizations were performed on the derived polysaccharides. Enzyme?linked immunosorbent assay and reverse transcription?polymerase chain reaction analyses demonstrated that K5?OS2 stimulated murine RAW264.7 macrophage cells to release TNF?α and IL?1β proinflammatory cytokines. K5?OS2 also induced the expression of inducible nitric oxide synthase iNOS， which is responsible for the production of nitric oxide. In addition， K5?OS2 markedly induced macrophage?mediated cytotoxicity against cancer cells and promoted the phagocytic activity of the RAW264.7 cells. Therefore， K5?OS2 activated macrophages and acted as a potent immunomodulator. Observations of the present study also indicated that sulfation modification enhanced the immune?enhancing activity of K5PS， and that the high sulfation in the O?position of K5PS may be required for the immunomodulatory activities of the Escherichia coli K5 capsular polysaccharide.