Arteriosclerosis is an important age-dependent disease that encompasses atherosclerosis, in-stent restenosis, autologous bypass grafting and transplant arteriosclerosis. Vascular smooth muscle (vSMC)-like accumulation is a key event leading to intimal-medial thickening (IMT), vessel remodelling and an important marker of subclinical disease. Vascular stem cell progeny in addition to de-differentiated SMC and/or SMC derived from endothelial-mesenchymal transition (EndoMT) are all reported to contribute to IMT as they become activated/dysfunctional, differentiate down vascular and myeloid lineages and subsequently dictate, in-part, vessel remodelling. In this study we examined the multipotent potential of a specialised population of rat adult resident multipotent vascular stem cells (rMVSC) located within the vessel wall and their capability to differentiate down both myogenic (muscle) and vasculogenic (endothelial) lineages when given the appropriate stimulus. Using Sca1-eGFP transgenic mice in vivo, there was a significant increase in the number of Sca1+ cells within the intima of the left carotid artery (LCA) following partial carotid artery ligation-induced injury after 3 days that co-localised with endothelial nitric oxide synthase (eNOS) and CD31+ positive cells, when compared to the sham-operated control vessels and the contralateral right carotid artery (RCA). The number of Sca1-eGFP+ cells significantly increased over time within the adventitial, medial and neointimal layers following ligation-induced injury after 7 and 14 days, respectively. In vitro, S100β/Sca1+ rat MVSCs cultured in vasculogenic inductive media for 7 days underwent differentiation to an endothelial cell phenotype characterised by the appearance of a cobblestone morphology and increased eNOS expression. In contrast, MVSC exposed to media supplemented with TGF-β1 for 7 days underwent myogenic differentiation to SMC-like cell. These data suggest that resident S100β/Sca1+ MVSCs are capable of both myogenic and vasculogenic differentiation depending on the inductive stimulus and may contribute in part to intimal medial thickening (IMT) and endothelial regeneration following injury in vivo.