Rolled-up morphology of bilayer sheets in a self-assembled glycolipid nanotube (LNT) in water was carefully examined by using a cryogenic transmission electron microscope (cryo-TEM) with a rapid-freezing specimen-preparation technique. The LNTs were obtained under a series of self-assembly conditions: boiling of an aqueous dispersion of glycolipid N-(11-cis-octadecenoyl)-beta-D-glucopyranosylamine, subsequent gradual cooling, and incubation at room temperature for several days. Cryo-TEM images revealed that the LNT walls consist of a multilayer structure with interlayer distance of about 4.7 nm. These layers correspond to constituent lipid bilayers. From the result of precise cryo-TEM observations and analyses, we confirmed the rolled-up morphology of the lipid bilayer sheets in a complete self-assembled glycolipid nanotube.