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The M3 subtype of muscarinic acetylcholine receptors (M3-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 M3-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M3-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 M3-mAChR. In H9c2 cells, the viability, intracellular free Ca2+ concentration ([Ca2+]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 M3-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly upregulated in ischemic heart tissue and the M3-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H2O2-induced H9c2 cell injuries measured by cells viability, [Ca2+]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M3-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M3-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 M3-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 M3-mAChR provides cardioprotection against myocardial ischemia injury. [ABSTRACT FROM AUTHOR]