The present study was designed to investigate the cardiac benefits of M 3 muscarinic receptor (M 3 -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 M 3 -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 M 3 -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, M 3 -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, M 3 -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 M 3 -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.