From the aqueous extract of fruit bodies of Flammulina velutipes, Flammulin, an antitumor substance, was purified to homogeneity judged by SDS-polyacrylamide gel electrophoresis. The purification procedure included ammonium sulfate precipitation, ethanol functionation, DEAE-Sephadex A50 anion-exchange chromatography, crystallization and recrystallization. Flammulin was crystallized by microdialysis against 12% polyethylene glycol 6000, in 20 mM sodium phosphate buffer (pH 7.0). The crystals were rhombohedron-shaped and easy to melt when the crystallization conditions were changed. With a molecular mass of 24000, Flammulin is characterized by its lack of methionine. Amino acid composition analysis showed that the acidic amino acids were in greater number than the basic amino acids, but the isoelectric point detected by analytical isoelectric focusing was at pH 8.9.