Hyperglycaemia increases the generation of reactive oxidants in blood vessels and is a major cause of endothelial dysfunction. A water-soluble selenium-containing sugar (1,4-Anhydro-4-seleno-d-talitol, SeTal) has potent antioxidant activity in vitro and is a promising treatment to accelerate wound healing in diabetic mice. One possible mechanism of SeTal action is a direct effect on blood vessels. Therefore, we tested the hypothesis that SeTal prevents endothelial dysfunction by scavenging reactive oxidants in isolated mouse aorta under conditions of acute oxidative stress induced by hyperglycaemia. Aortae were isolated from C57BL/6 male mice and mounted on a wire-myograph to assess vascular function. In the presence of a superoxide radical generator, pyrogallol, 300 μM and 1 mM of SeTal effectively prevented endothelial dysfunction compared to other selenium-containing compounds. In a second set of ex vivo experiments, mouse aortae were incubated for three days with either normal or high glucose, and co-incubated with SeTal at 37 °C in 5% CO2. High glucose significantly reduced the sensitivity to the endothelium-dependent agonist, acetylcholine (ACh), increased superoxide production and decreased basal nitric oxide (NO•) availability. SeTal (1 mM) co-treatment prevented high glucose-induced endothelial dysfunction and oxidative stress in the mouse aorta. The presence of a cyclooxygenase inhibitor, indomethacin significantly improved the sensitivity to ACh in high glucose-treated aortae, but had no effect in SeTal-treated aortae. Our data show that SeTal has potent antioxidant activity in isolated mouse aortae and prevents high glucose-induced endothelial dysfunction by decreasing superoxide levels, increasing basal NO• availability and normalising the contribution of vasoconstrictor prostanoids.