Hydroxysafflor yellow A (HSYA) was reported neuroprotective under several ischemic models in vivo. In this study, the direct effect of HSYA against oxygen-glucose deprivation (OGD) inducing acute neuronal injury and the underling mechanisms in vitro were investigated. Four-hour oxygen and glucose deprivation (OGD) followed by 20 h reperfusion (adding back oxygen and glucose, OGD-R) was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. HSYA (1, 10, and 100 μmol/l) was added to the cultures 30 min prior to the ischemic insult and was present during OGD and reoxygenation phases. The survival rate of PC12 cells was detected by MTT assay. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) activity were elevated by biochemical method. Hoechst 33258 staining and flow cytometric analysis were used to detect apoptosis; western blotting was used to detect the expression of Bcl-2, Bax, and Cytochrome C protein. The activity of caspase-3 was assessed by colorimetry. HSYA concentration-dependently attenuated neuronal damage with characteristics of increasing injured neuronal absorbance of MTT, decreasing cell apoptosis, and antagonizing decreases in SOD activity and increase in MDA level induced by OGD-R. Moreover, the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol and the consequent activation of caspase-3 were reversed by HSYA in a dose-dependent manner. These results suggest that apoptosis is an important characteristic of OGD-R-induced PC 12 death and that treatment of PC12 cells with HSYA can block OGD-R-induced apoptosis through suppression of intracellular oxidative stress and mitochondria dependent caspase cascade.