Objective: Previous studies have shown that mitochondrial division inhibitor 1 (mdivi-1) protects rat brain from ischemia/reperfusion (I/R) injury, but the precise mechanisms are unclear. This study aims to elucidate the effect of mdivi-1 on energy metabolism and neuronal apoptosis induced by I/R in vitro. Methods: Cultured hippocampal neurons from Wistar rats were randomly divided into four treatment groups: control (C), vehicle (V), I/R (I), and I/R plus mdivi-1 (M). Ischemia/reperfusion was induced by oxygen-glucose deprivation for 6 h followed by normoxic/normoglycemic reperfusion for 20 h. Neurons in the M group were pretreated with mdivi-1 for 40 min before I/R. Expression levels of the mdiv-1 target dynamin-related protein 1 and apoptosis regulators Bcl-2 and Bax were examined by Western blotting. Mitochondrial membrane potential () was measured by flow cytometry. Intracellular ATP content and the activities of mitochondrial complexes I−IV, Na+-K+-ATPase, and Ca2+-Mg2+-ATPase were measured by standard assays. Results: Compared to group I neurons, group M neurons exhibited markedly reduced Drp-1 and Bax expression, higher Bcl-2 expression, , and intracellular ATP, and elevated mitochondrial complex I−IV, Na+-K+-ATPase, and Ca2+-Mg2+-ATPase activities. Conclusion: Inhibition of mitochondrial fission significantly improved mitochondrial function and suppressed apoptotic signaling induced by I/R. [ABSTRACT FROM AUTHOR]