The optic nerve in two species of subterranean mole-rats (Spalacidae) has been examined at the ultrastructural level. The axial length of the eye and the diameter of the optic nerve are 1.9 mm and 52.5 μm in Spalax leucodon, and 0.7 mm and 80.8 μm in Spalax ehrenbergi, respectively. An anti-glial fibrillary acidic protein postembedding procedure was used to distinguish glial cell processes from axons. In both species, the optic nerve is composed exclusively of unmyelinated axons and a spatial distribution gradient according to the size or the density of fibers is lacking. The optic nerve of S. leucodon contains 1790 fibers ranging in diameter from 0.07–2.30 μm (mean = 0.57 μm), whereas in S. ehrenbergi, only 928 fibers, with diameters of 0.04–1.77 μm (mean = 0.53 μm) are observed. In S. ehrenbergi, a higher proportion of glial tissue is present and the fascicular organization of optic fibers is less obvious. Distribution gradients according to size frequency or density of fibers in the optic nerve are absent in both species. Comparison with other mammals suggests that although ocular regression in microphthalmic species is correlated with a significant decrease in the total number of optic fibers and the relative proportion of myelinated fibers, no difference in the absolute size range of unmyelinated axons is observed. The total absence of myelinated fibers in Spalax may be related to the subcutaneous location of the eyes. The unique presence of unmyelinated fibers in the optic nerve is discussed in relation to the possible conservation of a single class of W-like ganglion cells in the retina, in relation to photoperiodic perception.