Objective To study the anti-apoptosis effects of gentiopicroside in neonatal rat hippocampal neurons following oxygen-glucose deprivation and reperfusion injury and elucidated the related mechanisms. Methods The neonatal rat hippocampal neurons were pretreated with gentiopicroside 24h before exposed to oxygen-glucose deprivation and reperfusion injury were used as an in vitro model of ischemia and reperfusion. The neuron morphological change and apoptosis rate were evaluated by Hoechst 33342 staining. The nerve cell lactate dehydrogenase (LDH) leakage rate was detected by spectrophotometry. The expression levels of Caspase-3, Bax and Bcl-2 mRNA and protein were detected by RT-PCR and Western-blot assay respectively. Results Compared with the control group, oxygen-glucose deprivation and reperfusion injury model group could significantly enhance the apoptosis rate of nerve cells, increase the LDH leakage rate. Compared with the model group, pretreated with gentiopicroside (40, 20, 10mg/L) could significantly attenuated neuronal damage, with evidence of decreased neurons apoptosis rate and LDH leakage rate. Compare with the model group, pretreated with gentiopicroside (40 mg/L) could effectively down regulate the expression of Caspase-3 and Bax in mRNA and protein level, and up regulate the expression of Bcl-2 level. Conclusion Gentiopicroside preconditioning could prevent neurons from oxygen-glucose deprivation and reperfusion injury, and the mechanism may be mediated by up regulated Bcl-2 and down regulated Caspase-3 and Bax.