The present study was designed to investigate the cardiac benefits of M₃ muscarinic receptor (M₃-mAChR) overexpression and whether these effects are related to the regulation of the inward rectifying K⁺ channel by microRNA-1 (miR-1) in a conditional overexpression mouse model. A cardiac-specific M₃-mAChR transgenic mouse model was successfully established for the first time in this study using microinjection, and the overexpression was confirmed by both reverse transcriptase-polymerase chain reaction and Western blot techniques. We demonstrated that M₃-mAChR overexpression dramatically reduced the incidence of arrhythmias and decreased the mortality in a mouse model of myocardial ischemia-reperfusion (I/R). By using whole-cell patch techniques, M₃-mAChR overexpression significantly shortened the action potential duration and restored the membrane repolarization by increasing the inward rectifying K⁺ current. By using Western blot techniques, M₃-mAChR overexpression also rescued the expression of the inward rectifying K⁺ channel subunit Kir2.1 after myocardial I/R injury. This result was accompanied by suppression of upregulation miR-1. We conclude that M₃-mAChR overexpression reduced the incidence of arrhythmias and mortality after myocardial I/R by protecting the myocardium from ischemia in mice. This effect may be mediated by increasing the inward rectifying K⁺ current by downregulation of arrhythmogenic miR-1 expression, which might partially be a novel strategy for antiarrhythmias, leading to sudden cardiac death.