3'-Methoxypuerarin (3'-MOP) is an isoflavone extracted from radix puerariae. The aim of this study was to investigate the role and the mechanism of 3'-MOP in the protection of hippocampal neurons against cerebral ischemia/reperfusion (I/R) injury in rats. I/R injury was induced by a modified fourvessel occlusion model. Rats were randomly divided into an I/R group, an I/R + 3'-MOP group and a control group. Histological changes in the neurons of the hippocampal CA1 region were observed with hematoxylin and eosine (H&E) staining. The apoptotic neurons in the hippocampal CA1 area were counted with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The results showed that compared with the I/R group, 3'-MOP increased the number of surviving neurons in the hippocampal CA1 region (P<0.001) and markedly reduced the number of apoptotic pyramidal neurons (P<0.001) after I/R injury. In conclusion, 3'-MOP can protect hippocampal neurons against I/R injury by inhibiting apoptosis.