1,2,5,6-Dianhydro-3,4-diacetyl-galactitol (DADAG), an alkylating sugar alcohol derivative, has been shown effective against tumor growth. In this research, we explored the effect of DADAG on angiogenesis in chick chorioallantoic membrane (CAM) model and on the proliferation and migration of human umbilical vein endothelial cells (HUVECs). We also studied the possible mechanism of the anti-angiogenesis effect of DADAG. The results showed that DADAG (100, 500 and 1000μnol/L) inhibited angiogenesis in CAM model dose-dependently. Sulforhodamine B (SRB) assay indicated that DADAG (45, 90, 135, 180 and 225 μmol/L) suppressed HUVECs proliferation in a dose-dependent and time-dependent manner. High Content Screening (HCS, Cellomics) assay, in which the influence of cell proliferation on migration could be excluded, indicated that DADAG (45, 135 and 225 ~xmol/L) directly inhibited the motility ofHUVECs. Immunofiuorescence assay suggested that DADAG inhibited angiogenesis possibly by decreasing vascular endothelial growth factor (VEGF) expression in HUVECs. Our findings reveal that DADAG show anti-angiogenic activity in vivo and in vitro, which is related to the downregulation of VEGF expression in endothelial cells.