Diabetes is one of high risk factors for cardiovascular diseases, including atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to hypertension in diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were monitored by western blot. Endothelial function was determined by organ bath. High glucose (HG) or thromboxane A2 mimetic U46619 significantly reduced the levels of p-eNOS and p-Akt in cultured HUVECs, which were reversed by inhibition of TPr. HG/U46619-induced reductions of p-eNOS and p-Akt were accompanied with increases of total and phosphorylated tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). PTEN siRNA restored Akt-eNOS signaling in cells treated with HG. In rats, streptozotocin-induced hyperglycemia was associated with aortic PTEN upregulation and reductions of p-Akt and p-eNOS. TPr antagonist SQ29548 ablated these alterations and reduced blood pressure in rats with DOCA-induced hypertensive. In conclusion, hyperglycemia activates thromboxane A2 receptor to augment DOCA-induced high blood pressure in rats via the PTEN-Akt-eNOS signaling.