Nicotinic acetylcholine receptor (nAChR) clustering is a key event in the synaptogenesis of the neuromuscular junction (NMJ) for the efficient transmission of neural signals from motor neurons to skeletal muscle. The microphthalmic mouse (mi/mi) with a mutation in the mitf gene cannot perform occlusion, because its teeth do not erupt. The present study attempted to elucidate the contribution of occlusion to the clustering of nAChR in the NMJ of the masseter, with mi/mi as a model system. In mice at 1 week of age, no significant change in the fragmentation or volume of the nAChR cluster was observed in either the masseter or gastrocnemius between breast-fed +/+ and mi/mi. In mice at 4 and 12 weeks of age, after the occlusion emerged in the +/+, excessive fragmentation and volume decline in the nAChR cluster were observed in the masseter of mi/mi fed a powdered diet compared with +/+ fed a pellet or powdered diet, whereas, in the gastrocnemius, no such differences were observed between the 2 strains. These results indicate abnormal formation of the nAChR cluster in the NMJ of the masseter of mi/mi, suggesting that occlusion is essential for the normal progress of nAChR clustering in the NMJ of the masseter.