Paeoniflorin (PF) is a compound isolated from Paeoniae Radix. PF ameliorated brain ischemia on middle cerebral artery occlusion (MCAO) model in rats which was probably mediated by activating adenosine A1 receptor (A1R) to exert its neuroprotective effect. The aim of this study was to investigate the molecular mechanism underlying the neuroprotective effect of PF pretreatment on primary cultured cortical neurons exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). Primary cultured cortical neurons of rats were subjected to OGD/R injury and treated with PF and the survivability of neurons was determined by MTT assay. Phosphorylation of the proteins in the signaling pathways involved in the PF effects was evaluated by Western Blot or immunoprecipitation (IP). Receptor interactions were identified by co-IP and immunofluorescence staining. PF, with concentrations ranging from 10nM to 1μM, was applied to promote the survival of cortical neurons suffered from OGD/R injury. PF phosphorylated Akt and ERK1/2 that contributed to improve the neurons viability. On HEK293 cells stably transfected with A1R (HEK293/A1R), PF induced Akt and ERK1/2 phosphorylation mediated by A1R transactivation of EGFR. Furthermore, both Matrix Metalloprotease (MMP) and Src kinase were involved in signaling pathways regulated by PF. Our results showed that PF promoted neurons survival and relied on Akt and ERK1/2 phosphorylation which was mediated by A1R transactivation of EGFR.