The neuromuscular synaptogenesis is regulated by two interdependent effects evoked by an essential synaptic organizing protein, agrin, and the neuromuscular transmitter, acetylcholine (ACh). Agrin induces acetylcholine receptor (AChR) clusters through MuSK / rapsyn pathway, whereas ACh disperses the clusters in a Cdk5-dependent manner. 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. The present study uncovers a role of calpain interaction with rapsyn in regulating postsynaptic differentiation at the neuromuscular junction. We found that calpain, a calcium-dependent protease, was activated by the cholinergic stimulation and involved in the Cdk5-dependent dispersal of AChR clusters. Inhibition or down-regulation of calpain increased AChR cluster stability in cultured myotubes. Interestingly, the AChR-associated protein rapsyn interacted with calpain in an agrin dependent manner, and this interaction inhibited the protease activity of calpain. Down-regulation of rapsyn increased the activity of calpain. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via over-expressing calpastatin, an endogenous calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and agrin stabilizes AChR clusters by suppressing calpain activity through rapsyn-calpain interaction.