Objective Proper synaptogenesis and synaptic maturation are required for accurate neuronal network formation and brain function.Many studies in vitro have indicated that Neuroligin, which interacts with Neurexin, forming a transynaptic contacts, is required for synaptogenesis and maturation of post synapse.Due to confusing splicing isforms and redundancy roles of multiple neuroligin genes in mammal, the precise role of each neuroligin gene was not elucidated yet.Previous studies have indicated Drosophila neuroligin 1 is only expressing on post synapse at NMJ, involved in synaptic growth and postsynaptic differentiation, whereas neuroligin 2 expresses not only on the post synapse at NMJ but also on the CNS and it is required for synaptic growth and both pre-and postsynaptic maturation.In present study, we take the advantage of genetic manipulation and powerful tools in Drosophila to dissect the function of Drosophila neuroligin3 in vivo.Methods The Drosophila neuroligin3 mutants were identified by immunostaining and immunoblotting.The expression pattern of Drosophila neuroligin3 and the morphology of NMJ were measured by immunostaining.The function of synapse was analyzed by electrophysiological recording.Results Drosophila neuroligin3 is a new member of neuroligin gene family in Drosophila, which is mainly expressed in CNS neuropile and type Ⅰ but not type Ⅱ boutons.Drosophila neuroligin3 mutants show a decrease in EJP amplitude and mEJP frequency.Interestingly, Drosophila neuroligin3 mutants show a decrease in type Is bouton number without affecting type Ⅰb bouton number.Further, glutamate receptor Ⅱ B recruitment is defect in Drosophila neuroligin3 mutants.Conclusions As a new member of Drosophila neuroligin gene family, neuroligin3 may be not involved in synaptogenesis but is required for glutamate receptor recruitment, especially the glutamate receptor Ⅱ B.