Endothelial glycocalyx refers the proteoglycan or glycoproteins layer of luminal side of vessel wall. It has several functions in regulating vessel permeability, sensing shear stress, and preventing from unnecessary leukocyte adhesion. Until now, assessment to cerebral endothelial glycocalyx has only performed in ex vivo studies using electron microscope and confocal microscope. However, in vitro studies need additional chemical steps such as fixation or dehydration that presumably damage the glycocalyx layer. Thus, to characterize the proper structure and function of cerebral endothelial glycocalyx, it is important to visualize it in in vivo system. Here I visualize and quantify cerebral endothelial glycocalyx in vivo by tagging with WGA-lectin under two-photon laser scanning microscope. Cerebral arteries and capillaries have intact endothelial glyococalyx but not in veins or venules. I quantified the thickness of endothelial glycocalyx layer at pial, penetrating arteries and capillary. In addition, the cerebral endothelial glycocalyx was imaged and quantified in type II diabetes model. The cerebral endothelial glycocalyx was degraded in diabetes mouse. In addition, the vessel density was increased in diabetes mouse. To test whether these changes are related with hemodynamic response, intrinsic optical signal imaging was conducted. However, only increasing trend of response duration was observed but not significant. These results suggest that distribution of cerebral endothelial glycocalyx reflects the functional inhomogeneity of the vessel type.