3,3'-Diindolylmethane (DIM), an indole derivative produced on consumption of broccoli and other cruciferous vegetables, has been shown to have multiple anticancer effects in both in vivo and in vitro models. The present study was carried out to clarify the mechanism of DIM's antiangiogenic activity. We found that DIM can inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation and DNA synthesis in human umbilical vascular endothelial cells (HUVECs). Consistent with this inhibition, VEGF-induced extracellular signal-regulated kinase (ERK1/2) phosphorylation was greatly reduced. However, VEGF receptor phosphorylation induced by VEGF was not affected by DIM, indicating that DIM does not exert a direct and specific effect on the tyrosine kinase activity of this receptor. Further studies showed that DIM had a similar inhibitory effect on ERK1/2 phosphorylation induced by a variety of growth factors. Furthermore, Ras-GTP content, which dramatically increased after HUVECs were challenged by either individual growth factors or serum, was reduced by approximately 80% with 25 muM DIM treatment, which in turn resulted in the reduced activities of Raf and MEK, culminating in the drop of ERK1/2 activation. Overexpression of constitutively active GTPase mutant, Ras G12V, in HUVECs reversed the inhibitory effect of DIM on ERK1/2 activation. In a rodent Matrigel plug model, the presence of DIM strongly reduced VEGF-induced neovascularization, indicating that DIM is active in vivo. These data provide evidence that DIM inhibits Ras signaling induced by VEGF and other growth factors, which interferes with its downstream biological effects necessary for angiogenesis.