The ubiquitin–proteasome pathway has been implicated in synaptic development and plasticity. However, mechanisms by which ubiquitination contributes to precise and dynamic control of synaptic development and plasticity are poorly understood. We have identified a PDZ domain containing RING finger 3 (PDZRN3) as a synapse-associated E3 ubiquitin ligase and have demonstrated that it regulates the surface expression of muscle-specific receptor tyrosine kinase (MuSK), the key organizer of postsynaptic development at the mammalian neuromuscular junction. PDZRN3 binds to MuSK and promotes its ubiquitination. Regulation of cell surface levels of MuSK by PDZRN3 requires the ubiquitin ligase domain and is mediated by accelerated endocytosis. Gain- and loss-of-function studies in cultured myotubes show that regulation of MuSK by PDZRN3 plays an important role in MuSK-mediated nicotinic acetylcholine receptor clustering. Furthermore, overexpression of PDZRN3 in skeletal muscle of transgenic mice perturbs the growth and maturation of the neuromuscular junction. These results identify a synapse-associated E3 ubiquitin ligase as an important regulator of MuSK signaling.