The present study examined how the existence of glycocalyx layer (GLY) could influence the wall shear stress (WSS) and nitric oxide (NO) bioavailability in small arterioles. It was accomplished by considering GLY in determination of WSS, and its consequential effect on NO diffusion was predicted by a time-dependent computational model. The results showed that the WSS with consideration of GLY was not significantly different from that without the consideration. Although the difference was not statistically significant, the mean WSS with consideration of GLY was slightly greater than that without the consideration. The corresponding computational simulation to the existence of GLY showed no significant effect of GLY consideration on the peak NO concentration in the endothelium. In addition, no significant difference was found in sGC activation between with and without consideration of GLY. The above findings suggest that small changes in WSS due to the GLY existence may not significantly influence the NO transport or sGC activation in arterioles.