Hyperglycemia is associated with serious microvascular complications in diabetes mellitus. We hypothesized that under high glucose concentrations, activation of the hexosamine pathway leads to protein O-linked-N-acetylglucosamine (O-GlcNAc) formation which interferes with nitric oxide (NO)-dependent arteriolar dilation. Thus, diameter changes of skeletal muscle arterioles (diameter: ~ 160 μm) isolated from healthy, male Wistar rats were investigated after exposure to normal glucose (NG, 5.5 mM) or high glucose (HG, 30 mM for 2 hours) concentrations. In arterioles exposed to HG concentration, dilations to histamine were markedly reduced compared to those of under NG condition (max: −6±6% and 69±9%, respectively). Inhibition of NO synthesis with L-NAME reduced histamine-induced dilations in NG arterioles, but it had no effect on microvessels exposed to HG. Dilations to the NO donor, sodium nitroprusside were similar in the two groups of vessels. In the presence of the inhibitor of hexosamine pathway, azaserine histamine-induced dilations were significantly augmented and diminished by additional administration of L-NAME in arterioles exposed to HG concentrations (max: 67±2%). Moreover, under NG condition, exposure of vessels to glucosamine (5 mM, for 2 hours) resulted in reduced histamine-induced arteriolar dilations (max: 26±3%). Upon HG and glucosamine treatment an enhanced O-GlcNAc formation of endothelial NO synthase (eNOS) was detected by Western blots and immunoprecipitation. These findings indicate that high glucose concentration may lead to O-GlcNAc formation of eNOS, which impairs histamine-induced, NO-mediated arteriolar dilations. We propose that interfering with the hexosamine pathway may prevent microvascular complications in diabetes mellitus. On the other hand, our data suggest caveat for glucosamine supplementation to treat osteoarthritis, since it may impair microvascular function.