During the development of vertebrate neuromuscular junction (NMJ), agrin stabilizes, whereas acetylcholine (ACh) destabilizes AChR clusters, leading to the refinement of synaptic connections.The intracellular mechanism underlying this counteractive interaction is not completely understood.Here we show that caspase-3, the effector protease involved in apoptosis, mediates elimination of AChR clusters.We found that caspase-3 was activated by cholinergic stimulation of cultured muscle cells without inducing cell apoptosis and this activation was prevented by agrin.Interestingly, inhibition of caspase-3 attenuated ACh agonist-induced dispersion of AChR clusters.Furthermore, we identified Dishevelled 1 (Dvl1), a Wnt signaling protein involved in AChR clustering, as the substrate of caspase-3.Specific blockade of Dvl1 cleavage also prevented induced dispersion of AChR clusters.Finally, inhibition or genetic ablation of caspase-3 resulted in stabilization of aneural AChR clusters.Thus, caspase-3 plays an important role in the elimination of postsynaptic structures during the development of NMJs.