This study was performed to demonstrate the effects of deacetylated chitohexaose (hexamer) separated from a chitooligosaccharide (COS) mixture on tumor angiogenesis and its mechanism of action. Five fractions from dimer to hexamer were separated by a linear gradient solution of HCl on a cation-exchange resin. Then HCl was removed from the fractions by a charcoal column. The purity of the five fractions was analyzed by HPLC and the molecular masses were analyzed by MALDI-TOFMS. The hexamer expressed an inhibitory influence on CAM angiogenesis in a dose-dependent manner at concentrations of 6.25-50microg/egg. On further investigation, we found that the hexamer had no toxic effect on normal ECV304 cells, but could inhibit the proliferation and migration of tumor-induced ECV304 cells in a dose-dependent manner. The mechanism was demonstrated through the detection of mRNA expression of VEGF, MMP-9, TIMP-1, TIMP-2, and uPA by RT-PCR, which showed that the hexamer down-regulated the VEGF and uPA mRNA expressions in ECV304 cells, but up-regulated the TIMP-1 mRNA expression.