Accumulating evidence suggests that Che-1 is a strong antiapoptotic protein and can protect cells against various insults. However, whether Che-1 is involved in the protection of neurons against cerebral ischemia/reperfusion injury remains unclear. In this study, we aimed to investigate the potential role of Che-1 in regulating cerebral ischemia/reperfusion injury-induced neuronal injury using the oxygen–glucose deprivation and reoxygenation (OGD/R) model in vitro. We found that Che-1 expression was induced in neurons following OGD/R treatment. Functional experiments showed that Che-1 knockdown aggravated OGD/R-induced neuronal apoptosis. In contrast, Che-1 overexpression exerted a protective effect against OGD/R-induced neuronal apoptosis. Moreover, our results showed that the protective effect of Che-1 was associated with the inhibition of p53-mediated proapoptotic genes, including Puma, Noxa, and Bax. In addition, we showed that Che-1 impeded the transcript activity of p53 toward apoptosis. Taken together, our results indicate that Che-1 alleviates OGD/R-induced neuronal apoptosis in-vitro through inhibition of p53-mediated proapoptotic signaling. Our study suggests that Che-1 may serve as a potential therapeutic target for the treatment of cerebral ischemic/reperfusion injury in vivo.