Objective: Reperfusion injury accounts for ~50% of myocardial infarct size, and meaningful clinical therapies targeting this do not exist. We have shown that HDAC inhibition-enhanced cardiomyocyte autophagy blunts ischemia/reperfusion (I/R) injury given at the time of reperfusion. However, as HDAC inhibition may have off-target effects, we set out to test whether augmentation of autophagy protects myocardium through maintenance of mitochondrial homeostasis and reduction of oxidative stress during reperfusion injury. Methods: 10-week-old, wild-type, C57BL6 mice were randomized into 3 groups: vehicle control, or exposed to a Tat-Scrambled (TS) peptide, or a Tat-Beclin (TB, autophagy-inducing molecule) peptide. Each group was subjected to I/R surgery (45min coronary ligation, 24h reperfusion). Infarct size, systolic function, and mitochondrial dynamics were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were exposed to TB during simulated ischemia/reperfusion injury. ATG7 knockout (ATG7 KO) mice and ATG7 knockdown by siRNA in NRVMs was used to evaluate the role of autophagy. Results: TB treatment at reperfusion reduced infarct size by 20.1% (n=23, p<0.05) and improved systolic function (n=11, p<0.05). Improvement correlated with increased autophagic flux in the border zone with less oxidative stress. ATG7 KO mice did not manifest TB-promoted cardioprotection during I/R. TB increased mtDNA content in the border zone (n=10, p<0.05). In NRVMs subjected to I/R, TB reduced cell death by 41% (n=12, p<0.001), reduced I/R-induced mtDNA damage, and increased mtDNA content by >60% (n=3, p<0.05). Moreover, TB promoted expression of the gene coding for PGC-1α, which controls mitochondrial biogenesis, in the border zone (n=10, p<0.05) and in NRVMs subjected to I/R (n=3, p<0.05), along with expression of the mitochondrial dynamics genes Drp1, Fis1 and MFN1 / 2 (n=9, p<0.05). Conversely, ATG7 knockdown in NRVMs abolished these beneficent effects of TB on mitochondria. Conclusions: Autophagy is a sufficient and essential process to mitigate reperfusion injury through maintenance of mitochondrial homeostasis. Augmentation of autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in myocardial infarction