Abstract: A new fraction ofFlammulina velutipes polysaccharide 1 (FVP1) was separated and purified from Flammulina velutipes mycelium with several processes.Its structural features and bioactivities in vitro were also detected.The molecular mass of FVP1 was determined by HPGPC；the monosaccharide composition was identified according to the result of gas chromatography (GC)；the glycosidic bonds between monosaccharides were analyzed by periodic acid oxidation-Smith degradation and methylation analysis.Fluorescamine was bound with FVP1 and fluorescent activated cell sorting (FACS) was used to analyze the binding of FVP1-FLA to peritoneal macrophages.The molecular mass is about 140 kD.FVP1 consists of mannose (Man),glucose (Glc),galactose(Gal),fucose(Fuc) and rhomnose (Rha) in a molar ratio of 2:4:5:1:1.It was found that FVP1 is a complicated polysaccharide with a main composition of 1,4-Glc,1,3-Gal,1,3-Fuc,1,6-Gal,1,3,6-Man,1,6-Glc,1,3,4-Rha by methylation analysis and gas chromatography-mass spectrometry (GC-MS).Finally,bioactivity analysis showed that FVP1 acted as an immunomodulator,which could bind to peritoneal macrophages and strongly stimulate them to produce NOin vitro.FVP1 was a highly branched polysaccharide with great potential as an immunomodulator.