Recent in vitro and in vivo studies have reported fluid shear stress-induced increases in endothelial layer hydraulic conductivity (Lp) that are mediated by increased production of nitric oxide (NO). Other recent studies have shown that NO induction by shear stress is mediated by the glycocalyx (GCX) that decorates the surface of endothelial cells. Here we find that selective depletion of the major components of the GCX with enzymes can block the shear stress-induced response of Lp. Consistent with their effects on NO production, we observe that heparinase and hyaluronidase block shear-induced increases in Lp. But chondroitinase, that does not suppress shear-induced NO production, does inhibit shear-induced Lp. A further surprise is that treatment with the general proteolytic enzyme, pronase, does not suppress the shear-Lp response. We also find that heparinase does not alter baseline Lp significantly, while chondroitinase, hyaluronidase and pronase increase it significantly.