Glaucoma is a leading cause of blindness, characterized by retinal ganglion cell (RGC) loss and optic nerve (ON) damage. Cumulative evidence suggests glial cell involvement in the degeneration of the ON and RGCs. We analyzed the contribution of microglial reactivity to early axoglial alterations of the ON in an induced model of ocular hypertension. For this purpose, vehicle or chondroitin sulfate (CS) were weekly injected into the eye anterior chamber from Wistar rats for different intervals. The amount of Brn3a(+) RGC significantly decreased in CS-injected eyes for 10 and 15 (but not 6) weeks. A reduction in anterograde transport of β-subunit cholera toxin was observed in the superior colliculus and the lateral geniculate nucleus contralateral to CS-injected eyes for 6 and 15 weeks. A disruption of cholera toxin β-subunit transport was observed at the proximal myelinated ON. A significant decrease in phosphorylated neurofilament heavy chain immunoreactivity, an increase in ionized calcium-binding adaptor molecule 1(+), ED1(+) (microglial markers), and glial fibrillary acidic protein (astrocytes) (+) area, and decreased luxol fast blue staining were observed in the ON at 6 and 15 weeks of ocular hypertension. Microglial reactivity involvement was examined through a daily treatment with minocycline (30 mg/kg, i.p.) for 2 weeks, after 4 weeks of ocular hypertension. Minocycline prevented the increase in ionized calcium-binding adaptor molecule 1(+), ED-1(+), and glial fibrillary acidic protein(+) area, the decrease in phosphorylated neurofilament heavy-chain immunoreactivity and luxol fast blue staining, and the deficit in anterograde transport induced by 6 weeks of ocular hypertension. Thus, targeting microglial reactivity might prevent early axoglial alterations in the glaucomatous ON. Cover Image for this issue: doi: 10.1111/jnc.13807.