Consumption of a high-fat high-fructose diet (western diet, [WD]) promotes vascular stiffness, a critical factor in the development of cardiovascular disease (CVD). Obese and diabetic women exhibit greater arterial stiffness than men, which contributes to the increased incidence of CVD in women. Further, high-fructose diets result in elevated plasma concentrations of uric acid via xanthine oxidase (XO) activation, and are also associated with vascular stiffness. In turn, uric acid levels lead to vascular stiffness and CVD in women. However, the mechanisms by which fructose contributes to vascular stiffness in females remain to be fully uncovered. We examined the role of XO inhibition on vascular stiffness in female C57BL/6J mice fed a WD or regular chow for 16 weeks. WD feeding resulted in increased arterial stiffness, measured by atomic force microscopy in aortic explants (16.19±1.72 vs 5.21±0.54 kPa, p<0.05), as well as abnormal aortic endothelium-dependent and independent vasomotor responses. XO inhibition using allopurinol (widely utilized in the clinical setting) significantly improved vasomotor responses and stiffness (16.9±0.50 vs 3.44± 0.50 kPa, p<0.05), while simultaneously lowering serum uric acid levels (0.55±0.98 vs 0.21±0.04 mg/dL, p<0.05). Also, allopurinol improved WD-induced markers of fibrosis and oxidative stress in aortic tissue, as analyzed by immunohistochemistry and transmission electronic microscopy. Collectively, these results demonstrate that XO inhibition protects against WD-induced vascular oxidative stress, fibrosis, impaired vasorelaxation and aortic stiffness in females. Further, excessive oxidative stress resulting from XO activation appears to play a key role in mediating vascular dysfunction induced by chronic exposure to WD in females.