Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell-extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, -silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of -silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with -silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein-transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with -silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.