In the present study, the underlying protective mechanism of melatonin on kainic acid (KA)-induced excitotoxicity was examined in the hippocampus of mice. KA, administered intracerebroventricularly (i.c.v.), induced marked neuronal cell death with concurrent microglial activation and subsequent induction of inducible nitric oxide synthase (iNOS) in the hippocampus. Histopathological analysis demonstrated that melatonin (10 mg/kg), administered 1 hr prior to KA, attenuated KA-induced death of pyramidal neurons in the CA3 region. Melatonin obviously suppressed KA-induced microglial activation and consequent iNOS expression that were determined by increased immunoreactivities of microglial marker OX-6 and iNOS, respectively. Increased phosphorylation of Akt in pyramidal neurons was observed as early as 2 hr after administration of melatonin. Further, melatonin resulted in increased expression of astroglial glial cell line-derived neurotrophic factor (GDNF), which started to appear approximately 6 hr after administration of melatonin. The results of the present study demonstrate that melatonin exerts its neuroprotective action against KA-induced excitotoxicity both through the activation of neuronal Akt and via the direct action on hippocampal neurons and through the increased expression of astroglial GDNF, which subsequently activates neuronal PI3K/Akt pathway. Therefore, the present study suggests that melatonin, pineal secretory product, is potentially useful in the treatment of acute brain pathologies associated with excitotoxic neuronal damage such as epilepsy, stroke, and traumatic brain injury.