White matter axons organize into fascicles that grow over long distances and traverse very diverse environments. The molecular mechanisms preserving this structure of white matter axonal tracts are not well known. Here, we used the optic nerve as a model and investigated the role of TAG-1, a cell adhesion molecule expressed by retinal axons. TAG-1 was first expressed in the embryonic retinal ganglion cells (RGCs) and later in the postnatal myelin-forming cells in the optic nerve. We describe the consequences of genetic loss of Tag-1 on the developing and adult retinogeniculate tract. Tag-1-null embryos display anomalies in the caliber of RGC axons, associated with an abnormal organization of the astroglial network in the optic nerve. The contralateral projections in the lateral geniculate nucleus are expanded postnatally. In the adult, Tag-1-null mice show a loss of RGC axons, with persistent abnormalities of axonal caliber and additional cytoskeleton and myelination defects. Therefore, TAG-1 is an essential regulator of the structure of RGC axons and their surrounding glial cells in the optic nerve.