This study was intended to further explain the mechanism of glutamate (Glu) mediated neurotoxicity. The concentrations of TCa and Ca2+ i were measured in 32 cortical neuron cultures, which were divided into four groups: normal control group (n = 8); 0.5 mmol/L Glu group (Glu group n = 8); 0.5 mmol/L Glu + 100 mmol/L Nimodipine group (antagonist I, n = 8); 0.5 mmol/L Glu + 12 mumol/L MK-801 group (antagonist II, n = 8). The results showed that TCa and Ca2+ i concentrations in Glu group were significantly higher than those in normal control group (P < 0.05); TCa and Ca2+i in both antagonist I and II groups were evidently lower than those in Glu group (P < 0.05); No difference was found between antagonist I and II groups. The results suggest that Glu neurotoxicity is due to the intracellular calcium overload, which may be from the pathway of voltage-dependent calcium channels (VDCCs) and NMDA receptor-operated channels (NROCs).