A reduction of the nitric oxide (NO) action in vascular smooth muscle cells (VSMC) could play a role in the vascular damage induced by the glycaemic excursions occurring in diabetic patients; in this study, we aimed to clarify whether a short-term incubation of cultured VSMC with high glucose reduces the NO ability to increase cGMP and the cGMP ability to phosphorylate VASP at Ser-239. We observed that a 180 min incubation of rat VSMC with 25 mmol/L glucose does not impair the NO-induced cGMP increase but reduces VASP phosphorylation in response to both NO and cGMP with a mechanism blunted by antioxidants. We further demonstrated that high glucose increases radical oxygen species (ROS) production and that this phenomenon is prevented by the PKC inhibitor chelerythrine and the NADPH oxidase inhibitor apocynin. The following sequence of events is supported by these results: (i) in VSMC high glucose activates PKC; (ii) PKC activates NADPH oxidase; (iii) NADPH oxidase induces oxidative stress; (iv) ROS impair the signalling of cGMP, which is involved in the antiatherogenic actions of NO. Thus, high glucose, via oxidative stress, can reduce the cardiovascular protection conferred by the NO/cGMP pathway via phosphorylation of the cytoskeleton protein VASP in VSMC.