Excitatory neurotoxicity has been implicated in many pathological situations and there is no effective treatment available. Humanin is a 24-aa peptide cloned from the brain of patients with Alzheimer's disease (AD). In the present study, excitatory toxicity was induced by N-methyl-D-aspartate (NMDA) in primarily cultured rat cortical neurons. MTT assessment, lactate dehydrogenase (LDH) release, and calcein staining were employed to evaluate the protective activity of humanin on NMDA induced toxicity. The results suggested that NMDA (100 μmol/L, 2.5 hr) triggered neuronal morphological changes, lactate dehydrogenase (LDH) release (166% of the control), reduction of cell viability (about 50% of the control), and the decrease of living cell density (about 50% of the control). When pretreated with humanin, the toxicity was suppressed. The living cells' density of humanin treated group was similar to that of control. The cell viability was attenuated dose-dependently (IC50 = 0.132 nmol/L). The LDH release was also neutralized in a dose-dependent manner. In addition, the intracellular Ca(2+) overloading triggered by NMDA reverted quickly and humanin could not inhibit it. These findings indicate that humanin can rescue cortical neurons from NMDA-induced toxicity in rat but not through interfering with NMDA receptor directly.