Agrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, and caspase-3, an effector caspase in apoptotic pathway, are activated by the cholinergic stimulation while inhibited by agrin. Inhibition of calpain and caspase-3 prevents CCh-induced dispersion of AChR clusters. Interestingly, the AChR-associated protein rapsyn interacted with and inhibited the protease activity of calpain. Agrin increased interaction of rapsyn with calpain. Disrupting the endogenous rapsyn / calpain interaction enhanced CCh-induced dispersion of AChR clusters in vitro. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain and caspase-3 participates in ACh-induced dispersion of AChR clusters, and agrin stabilizes AChR clusters by suppressing their activity.