Acute increases in cellular protein O-linked N-acetyl-glucosamine (OGlcNAc) modification (O-GlcNAcylation) have been shown to have protective effects in the heart and vasculature. We hypothesized that D-glucosamine (D-GlcN) and Thiamet-G, two agents that increase protein O-GlcNAcylation via different mechanisms, inhibit TNF-α-induced oxidative stress and vascular dysfunction by suppressing inducible nitric oxide (NO) synthase (iNOS) expression. Rat aortic rings were incubated for 3h at 37°C with D-GlcN or its osmotic control L-glucose (L-Glc) or with Thiamet-G or its vehicle control (H 2O) followed by the addition of TNF-α or vehicle (H 2O) for 21 h. After incubation, rings were mounted in a myograph to assess arterial reactivity. Twenty-four hours of incubation of aortic rings with TNF-α resulted in 1) a hypocontractility to 60 mM K + solution and phenylephrine, 2) blunted endothelium-dependent relaxation responses to ACh and substance P, and 3) unaltered relaxing response to the Ca 2+ ionophore A-23187 and the NO donor sodium nitroprusside compared with aortic rings cultured in the absence of TNF-α. D-GlcN and Thiamet-G pretreatment suppressed the TNF-α-induced hypocontractility and endothelial dysfunction. Total protein OGlcNAc levels were significantly higher in aortic segments treated with D-GlcN or Thiamet-G compared with controls. Expression of iNOS protein was increased in TNF-α-treated rings, and this was attenuated by pretreatment with either D-GlcN or Thiamet-G. Dense immunostaining for nitrotyrosylated proteins was detected in the endothelium and media of the aortic wall, suggesting enhanced peroxynitrite production by iNOS. These findings demonstrate that acute increases in protein O-GlcNAcylation prevent TNF-α-induced vascular dysfunction, at least in part, via suppression of iNOS expression. © 2012 the American Physiological Society.