An increase in the number of fragmented mitochondria contributes to the pathogenesis of ischemia-reperfusion (I/R) injury. Also, mitochondrial fission has shown an increase in obese condition. However, the cardioprotective roles of a mitochondrial fission inhibitor in obesity with cardiac I/R injury are unclear. We hypothesized that a fission inhibitor (Mdivi-1) reduces cardiac dysfunction during I/R injury in pre-diabetic rats. Male Wistar rats (n=40) were received a high-fat diet for 12 weeks to induce prediabetes. Then, rats underwent a 30-min coronary artery ligation was performed followed by reperfusion for 120 min. These I/R rats were given either: (1) vehicle or Mdivi-1 treatment at 3 time points relative to onset of ischemia: (2) pre-ischemia; (3) during ischemia; and (4) at onset of reperfusion. Cardiac function, myocardial infarct size, mitochondrial function and dynamic balance were determined. Interestingly, Mdivi-1 given at any time points effectively attenuated mitochondrial reactive oxygen species production, depolarization, swelling, and dynamic imbalance, resulting in reduced arrhythmias, myocardial cell death, infarct size and enhanced cardiac performance during I/R injury in pre-diabetic rats. Taken together, inhibition of mitochondrial fission effectively protected the heart against cardiac I/R injury regardless of the time of administration in pre-diabetic rats.Copyright © 2020 Elsevier Inc. All rights reserved.