Here we present the first evidence that autoantibodies against muscle-specific kinase (MuSK) can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG-like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin-independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin-independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin-independent AChR clustering. MuSK plays multiple roles in pre-patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. These mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.