Mitochondrial fission and fusion are essential for maintaining mitochondrial quality control. However, their role in stress resistance remains unknown. Since Dynamin-related protein 1 (Drp1) plays an essential role in mediating mitochondrial fission, we used cardiac-specific heterozygous Drp1 KO (Drp1-hetCKO) mice whose cardiac function is normal at 12 weeks of age. In order to evaluate the role of Drp1 in mediating stress resistance, 12-week-old Drp1-hetCKO and control (Ctr) mice were subjected to myocardial ischemia (30 min)/reperfusion (24 hours) (I/R). I/R injury increased the translocation of Drp1 from cytosol to mitochondria in Ctr mice, whereas this translocation was attenuated in Drp1-hetCKO mice.The infarct size/area at risk after I/R was significantly greater in Drp-1 hetero KO mice than in control mice (55.2 ± 3.0 vs. 40.2 ± 1.6%). Electron microscopic analyses showed that I/R significantly decreased mitochondrial mass and increased the number of autophagosomes containing mitochondria in control mice. However, these changes were significantly attenuated in Drp1-hetCKO mice, suggesting that endogenous Drp1 mediates mitochondrial fission and mitophagy after I/R (Relative mitochondrial mass, Ctr, baseline: 1.0±0.3, I/R: 0.7±0.4; Drp1-hetCKO, baseline: 1.8±0.3, I/R: 1.7±0.3). We also examined mitophagy using mitochondria-targeted Keima fluorescence. Keima has a bimodal excitation spectrum peaking at 440 and 560 nm, corresponding to the neutral and acidic pH states, respectively. The maturation of autophagosomes to autolysosomes can be monitored by measuring the fluorescence of mitochondria-targeted Keima. Fluorescent dots with high ratios of 560/440, indicating mitophagy, were significantly increased after GD in sh-scramble-transduced CMs (GD: 28.6±4.2, control 2.3±1.0 dots/cell, p<0.01) but not in sh-Drp1-transduced CMs (GD: 2.4±1.8, control 3.2±1.6 dots/cell, not significant), suggesting that GD stimulates mitophagy but that this is inhibited when Drp-1 is downregulated. These results suggest that endogenous Drp1 play an essential role in mediating mitochondrial fission and mitophagy, which in turn play an essential role in mediating myocardial protection in response to I/R.