Abstract Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre‐ and post‐synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single‐nucleus RNA sequencing (snRNA‐seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ‐constituting genes, Dok7 , Chrna1 , and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation ( Dok7 , Chrna1 , and Chrnd ) together with four representative β‐catenin regulators ( Amotl2 , Ptprk, Fam53b , and Tcf7l2 ). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where β‐catenin serves as a structural protein to organize the membrane‐anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where β‐catenin serves as a transcriptional coactivator in Wnt/β‐catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2 , but not of Amotl2 or Ptprk , impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA‐seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/β‐catenin signaling that regulate the formation of the embryonic NMJ. image