Objective To explore the regulation of Kelch‑like ECH‑associated protein 1 (Keap1)/nuclear factor‑E2‑related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway on mitochondrial fission after cerebral ischemia/reperfusion (I/R) injury. Methods The extracted primary hippocampal neurons of rats were divided into four groups according to the random number table method: a control group (group C), an oxygen‑glucose deprivation/reperfusion group (group OGD/R), an OGD/R+Nrf2 inhibitor group (group OGD/R+N) and an OGD/R+vehicle group (group OGD/R+V). The mitochondrial morphology was observed by electron microscopy. The expression of dynamin‑related protein 1 (Drp1), mitochondrial fission protein 1 (Fis1), Keap1 and Nrf2 were detected by Western blot. The nuclear translocation of Nrf2 protein was observed by immunofluorescence, and the apoptosis rate of each group was detected by flow cytometry. Results Compared with group C, group OGD/R presented reduced levels of Keap1 and increased levels of nuclear Nrf2 protein, Drp1 and Fis1; through immunofluorescence, the nuclear translocation of Nrf2 was found during OGD/R, with an increased apoptosis rate (P<0.05). Compared with group OGD/R, group OGD/R + N presented reduced levels of nuclear Nrf2 and increased levels of Keap1, with an increased apoptosis rate (P<0.05). There were no significant differences in the levels of Keap1, Drp1, Fis1 and nuclear Nrf2 protein and apoptosis rates between groups OGD/R+V and OGD/R (P>0.05). Conclusions During cerebral I/R, the Keap1/Nrf2/ARE signaling pathway can regulate mitochondrial fission, reduce apoptosis and relieve brain damage.