Abstract: The aim of this study was to investigate the effects of breviscapine on cultured rat hippocampal neuronal toxicity induced by glutamate. Primary hippocampal neurons were prepared from 2 day-old SD rats. After 8 days cultured In vitro, the cultures subjected to 30 min treatment of 0.1, 0.5 and 1.0 mmol·L^-1 L-glutamate, separately. Breviscapine (10, 20 and 40 μmol·L^-1) was added into the cultures during 30 min treatment of L-glutamate and for the following 24 h respectively. After 24 h of L-glutamate treatment, flow cytometric analysis of Annexin V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that L-glutamate dose-dependently induced hippocampal neuronal apoptosis and necrosis. In agreement with these results, RT-PCR experiments indicated a biphasic regulation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA after L-glutamate treatment, i.e up-regulation by 0.1 mmol·L^-1 L-glutamate and down-regulation by 0.5 and 1.0 mmol·L^-1 L-glutamate. However, breviscapine markedly reduced apoptosis and necrosis due to toxicity of 0.5 mmol·L^-1 L-glutamate. Compared with the vehicle-treated L-glutamate group, the apoptosis was reduced by 30.4% and 40.1%, and necrosis was reduced by 32.5% and 38.8%, after treatment by breviscapine of 20 and 40 μmol·L^-1. Meanwhile, breviscapine obviously reversed the down-regulation of XIAP expression induced by L-glutamate (up-regulation by 45.1% and 54.9% when compared with that of the vehicle-treated glutamate group). The results from the detection of confocal laser scanning microscopy with Fluo-3, a Ca²⁺ probe showed an obvious increase in intracellular Ca²⁺ during L-glutamate treatment; and breviscapine of 20 or 40 μmol·L^-1 significantly slowed down glutamate-induced Ca²⁺ influx and lowered the intracellular Ca²⁺ peak in hippocampal neurons (P<0.01). These results suggest that neuroprotective effect of breviscapine against glutamate excitotoxicity was associated with inhibition of the accumulation of intracellular Ca²⁺ and up-regulation of XIAP expression in hippocampal neurons.