The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (Argll) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting Argil with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against Argil. These results might be associated with increased L-arginine bioavailability. Collectively these results suggest that Argil may be a valuable target in age-dependent vascular diseases. © 2012 by the Korean Society for Biochemistry and Molecular Biology.