The mechanism of epilepsy-induced axonal impairment is poorly understood. N-methyl-D-aspartate receptors (NMDARs) play important roles in epilepsy and mediate structural and functional axonal impairment. GSK-3β and Cdk5 affect axons and are regulated by NMDARs, while their roles in epilepsy-induced axonal impairment are unclear. We demonstrated that axonal impairment is characterized by neurofilament heavy (NFH) reduction, amyloid precursor protein (APP) accumulation, and increased tau phosphorylation accompanied by a decrease of total tau in temporal lobe epilepsy (TLE) patients and pentylenetetrazol (PTZ)-kindled rats. Inhibiting NMDARs using memantine and ifenprodil alleviated NFH reduction and APP accumulation, decreased Cdk5 expression, and inhibited the activity of GSK-3β in the white matter of PTZ-kindled rats. Inhibiting GSK-3β and Cdk5 using lithium chloride and roscovitine also alleviated axonal impairment induced by PTZ. Therefore, axonal impairment in TLE may be mediated by NMDAR via GSK-3β and Cdk5. In addition, inhibiting either NMDARs or GSK-3β lowered the relative tau phosphorylation level by reversing the decrease of total tau without affecting phosphorylated tau S396 and T231. Meanwhile inhibiting Cdk5 lowered the tau phosphorylation level by reducing phosphorylated tau without affecting total tau, indicating a possible role of GSK-3β in NMDAR-mediated tau phosphorylation in epilepsy.