Objective To evaluate the role and the possible mechanisms of microRNA-25 (miR-25) in regulating oxygen-glucose deprivation (OGD)/reperfusion (OGDR)-induced cell apoptosis in cultured SH-SY5Y and IMR-32 cell. Methods The OGD model was constructed in human SH-SY5Y and IMR-32 cells to mimic I/R injury. Fragments mediated miR-25 overexpression, mi-R25 mimics, were synthesized and constructed into recombined lentivirus plasmid. Using lipofectamine, the plasmids were transfected into the cells. The cell viability and apoptosis were detected with MTT assay and TUNEL. The mRNA and protein expression of the target genes were detected using RT-PCR, Real-time PCR and Western blotting respectively. Results Compared with the normal group, miR-25 was down-regulated in OGDR model group with decreased cell viability and increased apoptosis. Meanwhile, mRNA and protein expression of Bax and Caspase-3 were increased, but with decreased expression of Bcl-2 (n=3, P<0.05). Compared with the OGDR group, cell viability was increased and the apoptosis was decreased in miR-25 transfection group, with decreased expression of Bax and Caspase-3, as well as increased expression of Bcl-2 (n=3, P<0.05). Conclusion Taken together, our result indicate that up-regulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through regulating Bax/Bcl-2 and Caspase-3 signaling pathway, which will provide a promising therapeutic target for cerebral ischemia/reperfusion injury.