Background and Purpose: Berberine, a natural isoquinoline alkaloid, exhibits a variety of pharmacological effects but the pharmacological targets and mechanisms are remained elusive. Here, we report a novel finding that berberine inhibits acetylcholine (ACh)-induced Ca2+ oscillations which may underlie the pathogenesis of the L-arginine-induced mouse model of acute pancreatitis (AP). Experimental approach: Patch-clamp recordings and confocal Ca2+ imaging were applied in acute dissociated pancreatic acinar cells prepared from CD1 mice to examine the effects of berberine on ACh-induced Ca2+ oscillations. The L-arginine-induced acute pancreatitis mouse model was used to evaluate the protective effects of berberine against pathological changes. Key Results: Whole-cell patch-clamp recordings showed that berberine (from 0.1 to 10 µM) reduced ACh-induced Ca2+ oscillations in a concentration-dependent manner, and this inhibition was also depended on ACh concentrations. The inhibitory effect of berberine neither occurred in intracellular targets nor extracellular cholecystokinin (CCK) receptors, chloride (Cl-) channels, and store-operated Ca2+ channels. Together, the results demonstrate that berberine directly inhibits M3 muscarinic receptors, which is further confirmed by the evidence of the interaction between berberine and M3 receptors in acinar cells. In a L-arginine AP model, berberine eliminates the ACh-induced Ca2+ oscillations, the elevation of pancreatic amylase and pulmonary myeloperoxidase, and improves acinar cell pathological injury. Conclusions and Implications: We provide novel evidence that berberine inhibits M3 receptors, in turn eliminates ACh-induced Ca2+ oscillations and L-arginine-enhanced Ca2+ signaling, which underlie the protective effects of berberine on pancreatic acinar cells against pathological changes in an acute pancreatitis model.