A defect in the gene for the lysosomal enzyme α-galactosidase A (Gla) results in globotriaosylceramide (Gb3) accumulation in Fabry disease and leads to premature death from cardiac and cerebrovascular events. However, gastrointestinal symptoms are often first observed during childhood in these patients, and are not well understood. In this study, we demonstrate an age-dependent microvasculopathy of the mesenteric artery (MA) in a murine model of Fabry disease (Gla-knockout) resulting from dysregulation of the vascular homeostatic enzyme, endothelial nitric oxide synthase (eNOS). The progressive accumulation of Gb3 in MA was confirmed by thin layer chromatographic analysis. A total absence of endothelium-dependent dilatation was present in MA of 8 month old Fabry mice, while suppression in acetylcholine-mediated vasodilatation was evident from 2 months of age. Endothelium-independent dilatation with sodium nitroprusside was normal compared with age-matched WT mice. The microvascular defect in the Fabry MA was endothelium-dependent and associated with a suppression of the active homodimer of eNOS. Phosphorylation of eNOS at the major activation site (Ser1179) was significantly down-regulated while phosphorylation of the major inhibitory site (Thr495) was remarkably enhanced in aged Fabry mouse MA. These profound alterations in eNOS bioavailability at 8 months were observed in parallel with high levels of 3-nitrotyrosine suggesting increased reactive oxygen species along with eNOS uncoupling in this vascular bed. Overall, the mesenteric microvessels in the setting of Fabry disease were observed to have an early and profound endothelial dysfunction associated with elevated reactive nitrogen species and decreased nitric oxide (NO) bioavailability.