Endothelial cell migration and tube formation in response to vascular endothelial growth factor (VEGF) play an important role in the process of angiogenesis. Recent data indicate that angiotensin type 2 (AT₂) receptor stimulation is antiangiogenic. Therefore, we studied the effect of angiotensin II (Ang II) on VEGF-induced migration and in vitro tube formation of human endothelial cells. Ang II inhibited VEGF-induced migration of EA.hy926 cells, human coronary artery (HCA) and human dermal microvascular (HDM) endothelial cells (ECs) as well as tube formation by HDMECs. The AT₂ receptor antagonist PD123,319 but not the AT₁ receptor antagonist losartan blocked the inhibitory effect of Ang II. The inhibitory effect of Ang II on VEGF-induced migration of endothelial cells was mimicked by the specific AT₂ receptor agonist CGP-42112A. The phosphorylation of Akt and its downstream effector endothelial NO synthase (eNOS) is pivotal to VEGF-induced angiogenesis. We therefore investigated the effect of Ang II on VEGF-induced Akt and eNOS phosphorylation. Ang II diminished the VEGF-induced phosphorylation of Akt and eNOS in endothelial cells, whereas the autophosphorylation of VEGF receptors was unaffected. CGP-42112A again mimicked and PD123,319 but not losartan blocked the inhibitory effect of Ang II. Treatment of endothelial cells with pertussis toxin (PTX) totally abolished the AT₂ receptor–mediated inhibition of VEGF-induced endothelial cell migration and blocked the inhibition of Akt and eNOS phosphorylation. In conclusion, this study indicates that AT₂ receptor stimulation inhibits VEGF-induced endothelial cell migration and tube formation via activation of a PTX-sensitive G protein. These findings may explain the reported antiangiogenic properties of the AT₂ receptor.