AbstractMetformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.