Myocardial ischemia/reperfusion (I/R) injury is one of the problems after coronary artery recanalization in patients with acute myocardial infarction, and the discovery of exosomes presents a broad potential for treating myocardial I/R injury. This work examined the function and regulatory mechanisms of exosomes produced from bone marrow mesenchymal stem cells (BMSCs-Exo) in myocardial I/R injury. Rats with I/R injuries had their myocardium directly injected with BMSCs-Exo. The outcomes demonstrated that cardiac function was enhanced and BMSCs-Exo dramatically decreased myocardial infarct size. Transcriptome sequencing was performed on heart tissues from the model and exosome-treated groups. GO and KEGG enrichment analyses revealed that exosomes might mitigate myocardial I/R damage via the AMPK/PGC-1α signaling pathway, confirmed by both in vitro and in vivo tests. The findings imply that compound C and sh-AMPK reverse the activation of PGC-1α and its downstream proteins and negate the protective effects of exosomes against oxidative stress and mitochondrial function in damaged cardiomyocytes. On the other hand, p-AMPK expression was unaffected by PGC-1α silencing. It was demonstrated that via activating the AMPK/PGC-1α signaling pathway, BMSCs-Exo might reduce oxidative stress and mitochondrial dysfunction in cardiomyocytes, thereby protecting against myocardial I/R damage.