Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is required for the formation and maintenance of neuromuscular junctions (NMJs). Although autoantibodies against MuSK have been demonstrated to cause myasthenia gravis (MG), the underlying pathogenic mechanism remains unclear because a major subclass of these antibodies is functionally monovalent. We investigated the pathogenic role of MuSK antibodies in the onset of MG in vivo and in vitro. Ultrastructural visualization of NMJs in paretic rabbits with MuSK antibodies indicated that postsynaptic membranes were preserved, despite a significant loss of complexity in the convoluted synaptic folds. In addition, an in vitro assay indicated that both divalent and monovalent antibodies from paretic rabbits could interfere with agrin-induced acetylcholine receptor (AChR) clustering in cultured myotubes. Furthermore, in the absence of agrin, divalent antibodies induced MuSK phosphorylation and accelerated downregulation of Dok-7, an essential intracellular MuSK binding protein, while monovalent antibodies inhibited agrin-induced phosphorylation of MuSK, thus demonstrating distinct molecular mechanisms underlying the MuSK dysfunction induced by these two types of antibodies. Taken together, these findings suggest that complement activation is not necessary for the MG onset and that both divalent and monovalent antibodies may cause MG in vivo by inducing MuSK dysfunction.