At the vertebrate neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is stimulated by motor neuron-derived glycoprotein Agrin and requires a number of intracellular signal or structural proteins, including AChR-associated scaffold protein Rapsyn. Here, we report a role of nuclear factor κB (NF-κB), a well known transcription factor involved in a variety of immune responses, in regulating AChR clustering at the NMJ. We found that downregulating the expression of RelA/p65 subunit of NF-κB or inhibiting NF-κB activity by overexpression of mutated form of IκB (inhibitor κB), which is resistant to proteolytic degradation and thus constitutively keeps NF-κB inactive in the cytoplasma, impeded the formation of AChR clusters in cultured C2C12 muscle cells stimulated by Agrin. In contrast, overexpression of RelA/p65 promoted AChR clustering. Furthermore, we investigated the mechanism by which NF-κB regulates AChR clustering. Interestingly, we found that downregulating the expression of RelA/p65 caused a marked reduction in the protein and mRNA level of Rapsyn and upregulation of RelA/p65 enhanced Rapsyn promoter activity. Mutation of NF-κB binding site on Rapsyn promoter prevented responsiveness to RelA/p65 regulation. Moreover, forced expression of Rapsyn in RelA/p65 downregulated muscle cells partially rescued AChR clusters, suggesting that NF-κB regulates AChR clustering, at least partially through the transcriptional regulation of Rapsyn. In line with this notion, genetic ablation of RelA/p65 selectively in the skeletal muscle caused a reduction of AChR density at the NMJ and a decrease in the level of Rapsyn. Thus, NF-κB signaling controls AChR clustering through transcriptional regulation of synaptic protein Rapsyn. Copyright © 2010 the authors.