The endothelial glycocalyx (GCX) mediates flow-induced nitric oxide release via heparan sulfate (HS), but the GCX structure is unclear and the specific HS core protein(s) involved in this mechanotransduction is unknown. Our study tests the hypotheses that flow regulates GCX thickness and organization and that the HS glypican-1 core protein mediates flow-induced activation of endothelial nitric oxide synthase (eNOS). Monolayers of bovine aortic and rat fat pad endothelial cells with intact GCX, enzymatically degraded HS, or RNA-silenced glypican-1 were exposed to 15 dyne/cm2 uniform shear stress for 3 hr. Confocal immunocytochemistry and cryotransmission electron microscopy revealed an unsheared GCX that was 2.5 to 4.0 μm thick. Sheared GCX was thicker, well organized and aligned perpendicular to the cell surface, while unsheared GCX was disorganized. Western blot and confocal microscopy demonstrated that when the GCX was intact, shear stress increased eNOS activation (ser1177 phosphorylation) and membrane localization, which were blocked by HS degradation. Flow-induced levels of activated eNOS were attenuated when glypican-1 was silenced. This work elucidates GCX structure and role in endothelial cell mechanotransduction.