Vesicle fusion contributes to the maintenance of synapses in the nervous system by mediating synaptic transmission, release of neurotrophic factors, and trafficking of membrane receptors. N-ethylmaleimide-sensitive factor (NSF) is indispensible for dissociation of the SNARE-complex following vesicle fusion. Although NSF function has been characterized extensively in vitro, the in vivo role of NSF in vertebrate synaptogenesis is relatively unexplored. Zebrafish possess two nsf genes, nsf and nsfb. Here, we examine the function of either Nsf or Nsfb in the pre- and postsynaptic cells of the zebrafish lateral line organ and demonstrate that Nsf, but not Nsfb, is required for maintenance of afferent synapses in hair cells. In addition to peripheral defects in nsf mutants, neurodegeneration of glutamatergic synapses in the central nervous system also occurs in the absence of Nsf function. Expression of an nsf transgene in a null background indicates that stabilization of synapses requires Nsf function in both hair cells and afferent neurons. To identify potential targets of Nsf-mediated fusion, we examined the expression of genes implicated in stabilizing synapses and found that transcripts for multiple genes including brain-derived neurotrophic factor (bdnf) were significantly reduced in nsf mutants. With regard to trafficking of BDNF, we observed a striking accumulation of BDNF in the neurites of nsf mutant afferent neurons. In addition, injection of recombinant BDNF protein partially rescued the degeneration of afferent synapses in nsf mutants. These results establish a role for Nsf in the maintenance of synaptic contacts between hair cells and afferent neurons, mediated in part via the secretion of trophic signaling factors.