Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90β (HSP90β) as a component in surface AChR clusters. The HSP90β-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90β activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90β was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90β in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.