Cell fate decisions within the vasculature are crucial to the pathogenesis of vascular diseases, including, arteriosclerosis, atherosclerosis and restenosis after angioplasty. Notch signalling is involved in regulating cell fate in vasculature development during embryogenesis resulting in altered cell fate decisions leading to vascular disease. The Notch signalling pathway is highly regulated by a number of mechanisms including glycosylation, a post-translational modification. The main objective of this research work was to define a putative role for N-glycosylation of Notch1 receptor in controlling resident vascular stem cell fate in vitro. Utilising ligand Jagged1 (Jag-1)-induced Notch signalling assay, Quantitative RT-PCR, immunocytochemistry, ectopic expression of Notch1 receptor, siRNA knockdown, pharmacological inhibition and enzyme linked lectin assay (ELLA), alterations in N-glycan decoration of the Notch1 receptor were assessed before the effects on Notch signalling and Notch ligand promotion of myogenic differentiation were evaluated. N-glycosylation of the Notch1 receptor was assessed using ELLA and confirmed the presence of N-glycans on the receptor, an effect that was abrogated following inhibition of glycosyltransferase activity with tunicamycin and Lunatic Fringe (Lfng) knockdown. Jag-1- induced Notch activation increased Notch target gene expression and promoted myogenic differentiation of bone-marrow derived mesenchymal stem cells and resident vascular stem cells. Selective knockdown of the Notch1 receptor in stem cells resulted in a decrease in Jag- 1 stimulated Hey1 Notch1 target gene expression concomitant with a reduction in myogenic differentiation due to decreased smooth muscle differentiation marker expression (Cnn1 protein level and Myh11 mRNA level). Inhibition of N-glycosylation with tunicamycin lead to a down regulation of smooth muscle differentiation marker, Myh11 independent of a reduction in Notch target gene expression. Lfng knockdown lead to a similar significant reduction in Jag-1 induced myogenic differentiation (reduced Myh11 mRNA expression). Collectively, these results suggest that N-glycosylation of Notch1 receptor is involved in Notch signalling leading to altered resident vascular stem cell fate.