<p>The key mechanism of myocardial ischemia–reperfusion injury (MIRI) is the oxidation reaction, and methyl rosmarinate (MR) has a strong antioxidant effect, but its role in MIRI has not been reported. About 3&#xa0;weeks after tail vein injection of AV-TGR5 and AV-sh-TGR5, MR was intraperitoneally injected, and the MIRI model was prepared by coronary artery ligation 3&#xa0;h later. The cardiac function of mice was evaluated by PowerLab system. The pathological damages of myocardial tissue were detected by kit, TCC, HE, Masson, and TUNEL staining. Oxidative stress and mitochondrial damage were assessed by TEM, DHE, and kits. The TGR5/AMPK axis protein levels were discovered using a Western blot. MR raised LVDP, + dp/dtmax, and − dp/dtmax values and significantly reduced LVEDP values, CK-MB and LDH contents, and myocardial infarct size. After MR intervention, the normal structure of myocardial fibers in mice was retained; inflammatory cell infiltration, myocardial fibrosis, and apoptosis were significantly reduced; and mitochondrial swelling and vacuolar degeneration and damage were improved. MR also significantly reduced ROS and MDA content and increased TGR5 and p-AMPK levels. After TGR5 overexpression, cardiac function was further enhanced; the myocardial pathological damage, oxidative stress, and mitochondrial damage were significantly improved; CK-MB and ROS contents were decreased again; and TGR5 and p-AMPK protein expressions were increased again. After TGR5 knockdown, the above indicators were significantly reversed. MR improves cardiac function in mice by activating the TGR5/AMPK signaling axis, reducing mitochondrial damage and oxidative stress, thereby alleviating MIRI.</p>