Axonal injury and dysfunction in white matter (WM) are caused by many neurologic diseases including ischemia. We characterized ischemic injury and the role of glutamate-mediated excitotoxicity in a purely myelinated WM tract, the mouse optic nerve (MON). For the first time, excitotoxic WM injury was directly correlated with glutamate release. Oxygen and glucose deprivation (OGD) caused duration-dependent loss of axon function in optic nerves from young adult mice. Protection of axon function required blockade of both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors, or removal of extracellular Ca(2+). Blockade of N-methyl-D-aspartate receptors did not preserve axon function. Curiously, even extended periods of direct exposure to glutamate or kainate or AMPA failed to induce axon dysfunction. Brief periods of OGD, however, caused glutamate receptor agonist exposure to become toxic, suggesting that ionic disruption enabled excitotoxic injury. Glutamate release, directly measured using quantitative high-performance liquid chromatography, occurred late during a 60-mins period of OGD and was due to reversal of the glutamate transporter. Brief periods of OGD (i.e., 15 mins) did not cause glutamate release and produced minimal injury. These results suggested that toxic glutamate accumulation during OGD followed the initial ionic changes mediating early loss of excitability. The onset of glutamate release was an important threshold event for irreversible ischemic injury. Regional differences appear to exist in the specific glutamate receptors that mediate WM ischemic injury. Therapy for ischemic WM injury must be designed accordingly.