Quercetin (QRN), one of the most abundant flavonoids in the human diet, is a known antioxidant and inhibitor of cancer cell cycle progression. Here, we provide the first evidence that QRN inhibits angiogenesis via a mechanism involving both suppression of endothelial nitric oxide synthase (eNOS) and early M-phase cell cycle arrest. Bovine aortic endothelial (BAE) cells were exposed to doses of up to 100 micromol/L QRN and assayed for eNOS activity and phosphorylation status. Phosphorylation of eNOS at Ser 617 (bovine sequence) is thought to occur in response to Akt stimulation and to be required for eNOS activity. Together with basal eNOS activity, eNOS phosphorylation at Ser 617 and Akt Ser 473 phosphorylation were dose dependently and concomitantly suppressed by QRN within 30 min. Furthermore, although the significant (P < 0.05) inhibitory effect of a single 100 micromol/L QRN dose on eNOS activity was overcome within approximately 24 h, chronic QRN exposures (24-48 h) led to early M-phase arrest and disruption of mitotic microtubule polymerization. In vivo, QRN administered i.p. to female Balb/C mice bearing both syngeneic mammary tumors and Matrigel implants suppressed angiogenesis as measured by endothelial cell immunohistochemistry and hemoglobin concentration. Taken together, these findings suggest a dual mechanism by which QRN suppresses endothelial cell proliferation, both acutely via inhibition of eNOS Ser 617 phosphorylation, and chronically via perturbation of mitotic microtubule polymerization. This novel mechanism of QRN in endothelial cells may in part explain its inhibitory action on angiogenesis and further discern a potential role of QRN as a chemopreventive agent.