We have proposed a long-term culture of C2C12 myotubes as a muscle atrophy model because the long-term culture induces myotubes atrophy via a mechanism similar to age-related muscular atrophy. The decrease in muscle strength due to aging involves not only the decrease in muscle mass but also the degeneration of the neuromuscular junction. In this study, we examined to reproduce the age-related degeneration of the neuromuscular junction using a muscle atrophy model. C2C12 myotubes and mouse embryonic stem cells-derived motor nerves were co-cultured in a co-culture medium, but the formation of acetylcholine receptor clusters at the neuromuscular junction hardly observed. Agrin-induced clustering of acetylcholine receptors in myotubes was also inhibited in this co-culture medium. However, on day 28 post-differentiation, suppression of agrin-induced clustering of acetylcholine receptors by co-culture medium was restored by replacement with medium for differentiation into myotubes. On day 56 post-differentiation, this restoration effect by replacement with the differentiation medium was attenuated. These results indicate that long-term culture of myotubes causes both atrophy of myotubes and failure of neuromuscular junction formations.