In tumor cells, the transcription factor NF-kappaB has been described to be antiapoptotic and proproliferative and involved in the production of angiogenic factors such as vascular endothelial growth factor. From these data, a protumorigenic role of NF-kappaB has emerged. Here, we examined in endothelial cells whether NF-kappaB signaling pathway is involved in mediating the angiostatic properties of angiogenesis inhibitors. The current report describes that biochemically unrelated agents with direct angiostatic effect induced NF-kappaB activation in endothelial cells. Our data showed that endostatin, anginex, angiostatin, and the 16-kDa N-terminal fragment of human prolactin induced NF-kappaB activation in endothelial cells in both cultured human endothelial cells and in vivo in a mouse tumor model. It was also found that NF-kappaB activity was required for the angiostatic activity, because inhibition of NF-kappaB in endothelial cells impaired the ability of angiostatic agents to block sprouting of endothelial cells and to overcome endothelial cell anergy. Therefore, activation of NF-kappaB in endothelial cells can result in an unexpected antitumor outcome. Based on these data, the current approach of systemic treatment with NF-kappaB inhibitors may therefore be revisited because NF-kappaB activation specifically targeted to endothelial cells might represent an efficient strategy for the treatment of cancer.