The endothelial glycocalyx plays important roles in mechanotransduction. We recently investigated the distribution and interaction of glycocalyx components on statically cultured endothelial cells. In the present study, we further explored the unknown organization of the glycocalyx during early exposure (first 30 min) to shear stress and tested the hypothesis that proteoglycans with glycosaminoglycans, which are localized in different lipid microdomains, respond distinctly to shear stress. During the initial 30 minutes of exposure to shear stress, the very early responses of the glycocalyx and membrane rafts were detected using confocal microscopy. We observed that heparan sulfate (HS) and glypican-1 clustered in the cell junctions. In contrast, chondroitin sulfate (CS), bound albumin and syndecan-1 did not move. The caveolae marker, caveolin-1, did not move indicating that caveolae are anchored sufficiently to resist shear stress during the 30 minutes of exposure. Shear stress induced significant changes in the distribution of ganglioside GM1 (a marker for membrane rafts labeled with cholera toxin B subunit - CTx-B). These data suggest that fluid shear stress induced the cell junctional clustering of lipid rafts with their anchored glypican-1 and associated HS. In contrast, the mobility of CS, transmembrane bound syndecan-1, and caveolae were constrained during exposure to shear stress. This study illuminates the role of changes in glycocalyx organization that underlie mechanisms of mechanotransduction.