The M 3 subtype of muscarinic acetylcholine receptors (M 3-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M 3-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M 3-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M 3-mAChR. In H9c2 cells, the viability, intracellular free Ca 2+ concentration ([Ca 2+]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M 3-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M 3-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H 2O 2-induced H9c2 cell injuries measured by cells viability, [Ca 2+]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M 3-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M 3-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M 3-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M 3-mAChR provides cardioprotection against myocardial ischemia injury.