Ischemic heart disease (IHD) is the most common cardiovascular disease and is the main cause of death and disability worldwide. Myocardial ischemia/reperfusion (MI/R) injury has been linked to IHD-induced cardiomyocytes apoptosis and tissue damage. Recently, it has been reported that carnosic acid (CA) may function as a potent antioxidant in liver ischemia/reperfusion (I/R). However, whether it regulates I/R in the heart remains unclear. Here, we elucidated the emerging role of CA in MI/R under diabetic myocardial conditions. Diabetes mellitus (DM) was induced in mice by consumption of a high-fat diet for 16 weeks. To create the I/R in mice, the left anterior descending coronary artery was occluded for 30 min, and then occlusion was released to reperfuse the heart for 3 or 24 h. In diabetic myocardial ischemia/reperfusion (DMI/R) mice, pre-treatment with CA suppressed the overgeneration of reactive oxygen species (ROS) and production of cytokine. Importantly, the activation of autophagy was significantly increased by CA treatment, as assessed by p62 degradation and LC3-II/LC3-I conversion, as well as by phosphorylation of AMPK alpha, Akt, and mTOR. Interestingly, all of the protective effects of CA were impeded by the treatment with chloroquine, which is an autophagy inhibitor. These studies suggest that CA prevents DMI/R injury via regulation of autophagy. In conclusion, our findings indicate that CA has potential as a novel therapeutic to prevent DMI/R injury.