A comparative study of scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging of the healthy human optic nerve was carried out to determine the similarities and the differences. In this study we compared the fine optic nerve structures as observed by SEM and AFM. The fibers of the right optic nerve of a 61-year-old man show different arrangements in transverse sections taken from the same individual 5 mm central to the optic canal and 5 mm peripheral to the optic chiasma; this difference can be recognized by light microscopy (LM), SEM, and AFM. AFM revealed such typical optic nerve fibers (taken from a point 5 mm central to the optic canal) with annular and longitudinal orientations, which were not visible by SEM in this form. By contrast, LM and SEM visualized other structures, such as pia mater and optic nerve fibers loosely arranged in bundles, none of which was visualized by AFM. The images, however, taken 5 mm peripheral from the optic chiasma show shapeless nerve fibers having a wavy course. Our results reveal that more detailed information on optic nerve morphology is obtained by exploiting the advantages of both SEM and AFM. These are the first SEM and AFM images of healthy human optic nerve fibers, containing clear representations of the three dimensions of the optic nerve.