Cardiovascular disease (CVD) is characterized by a thickening of blood vessel walls. This leads to the formation of a neointima, which occludes the lumen and restricts blood flow to vital organs. Mounting evidence suggests that resident stem cells in the vasculature may give rise to the smooth muscle-like cells which make up this neointima. In particular, a group of S100β+ cells, termed multipotent vascular stem cells (MVSCs), have been reported to contribute significantly to neointimal formation. Recent studies have shown that activation of Notch signalling, and Notch1 in particular, may be pivotal in this process as it is heavily involved in regulating stem cell fate and in directing myogenic differentiation. The Notch signalling pathway is highly regulated by a number of mechanisms including glycosylation. While the importance of O-linked glycosylation has been extensively described, the structures and roles of N-glycans on the Notch1 receptor have yet to be defined. The current study presents novel findings which confirm that activation of the Notch1 receptor by its ligand, Jag-1, stimulates myogenic differentiation in S100β+ MVSCs isolated from a mouse aortic arch. Furthermore, N-glycosylation intervention strategies, including small-molecule inhibition (using tunicamycin and 1-deoxymannojirimycin), siRNA knockdown of the GnT-III and Fut8 glycosyltransferases, and site-directed mutagenesis of N-glycosylation sites, revealed that this process is heavily regulated by N-glycans which reside at six consensus sequences along the extracellular domain of Notch1. A number of key oligosaccharides at these sites were found to contain bisecting or core fucosylated structures. Notch1 expression and trafficking analysis indicated that N-glycans at asparagine (N) 1241 and 1587 protect the receptor from accelerated degradation, while the oligosaccharide at N888 directly affects signal transduction. Conversely, N-linked glycans at the other three sites do not impact canonical Notch1 signalling but still play a role in Notch-mediated myogenic differentiation, possibly via a non-canonical Notch signalling pathway. The findings in the current study provide novel insights into the location and structure of N-glycans on the Notch1 receptor, as well as illuminating some of the likely mechanisms by which they regulate Notch signalling and myogenic differentiation in a CVD-relevant vascular stem cell population.