This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A(2) mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser(1177) and Akt at Ser(473). These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser(380)/Thr(382/383) PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G(i) family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser(380)/Thr(382/383) phosphorylation, and dephosphorylation of both Akt (at Ser(473)) and eNOS (at Ser(1177)). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.