5-LO inhibitor has been demonstrated to exert anti-inflammatory effect through inhibition of 5-lipoxygenase metabolism. However, its function in angiogenesis remains unknown. Here, we investigated the involvement of maxi-K channel activation in the antiangiogenic effect of zileuton, a well known 5-LO inhibitor. According to our data, pretreatment with zileuton efficiently inhibited proliferation and capillary tube formation in human umbilical vein endothelial cells (HUVEC). And these effects were reversed by iberiotoxin, a maxi-K channel blocker. Knockdown of maxi-K channel expression also reversed the antiangiogenic effects. We also found that zileuton exerted antiangiogenic effect via the inhibition of transcription factor Erg and phosphorylation of endothelial nitric-oxide synthase-Ser1179, one of the Erg target gene. Moreover, iberiotoxin and knockdown of maxi K channel significantly reversed not only the inhibition of Erg transcription but also phosphorylation of eNOS, suggesting maxi K channel as a key mediator of the zileuton actions. Our data strongly suggest that zileuton significantly inhibited VEGF-induced angiogenesis via Erg-mediated eNOS phosphorylation, followed by maxi-K channel opening.