Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A(2A) receptor (A(2A)R), but its role in EC ROS production remains unknown. In this study, we investigated the role of A(2A)R in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4-10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A(2A)R. These were accompanied by p47(phox), ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A(2A)R antagonist (SCH58261), or adenosine deaminase, or by knockdown of A(2A)R or Nox2 using specific siRNAs. Knockdown of A(2A)R, as determined by Western blotting, decreased Nox2 and p47(phox) expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A(2A)R knock-out mice. In conclusion, A(2A)R is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A(2A)R protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.