We aimed to test the hypothesis that an enhanced level of reactive oxygen species (ROS) is primarily responsible for the impairment of NO-mediated regulation of arteriolar wall shear stress (WSS) in hyperhomocysteinemia (HHcy). Thus, flow/WSS-induced dilations of pressurized gracilis muscle arterioles (basal diameter: ~170 µm) isolated from control (serum Hcy: 6±1 µmol/L), methionine diet-induced HHcy rats (4 weeks, serum Hcy: 30±6 µmol/L), and HHcy rats treated with vitamin-C, a known antioxidant (4 weeks, 150 mg/kg/day; serum Hcy: 32±10 µmol/L) were investigated. In vessels of HHcy rats, increases in intraluminal flow/WSS-induced dilations were converted to constrictions. The constrictions were unaffected by inhibition of NO synthesis by L-NAME. Vitamin-C treatment of HHcy rats reversed the WSS-induced arteriolar constrictions to L-NAME-sensitive dilations, but did not affect control responses. Similar changes in responses were obtained for the calcium ionophore A23187. In addition, diastolic and mean arterial blood pressure, and serum 8-isoprostane levels (a marker of in vivo oxidative stress) were significantly elevated in rats with HHcy, changes that were normalized by vitamin-C treatment. Taken together, our data show that in chronic HHcy long-term vitamin-C treatment by decreasing oxidative stress in vivo enhanced NO bioavailability, restored the regulation of shear stress in arterioles and normalized systemic blood pressure. Thus, our study provides evidence that oxidative stress is an important in vivo mechanism that is primarily responsible for the development of endothelial dysregulation of wall shear stress in HHcy.