Objective To determine if the Drosophila synaptic adhesion molecule neuroligin 4 (DNlg4) regulates the development of the larval neuromuscular synapse by interacting with the classic neuroligin (Nlg)-presynaptic ligand molecule Drosophila neurexin (DNrx). Methods Immunofluorescence staining and confocal microscopy were used to examine the morphology of the neuromuscular junction (NMJ) in Thrid-instar larvae in wild-type(WT), dnlg4 mutant, dnrx mutant, and dnlg4,dnrx double-mutant Drosophila. The length of the synaptic branches and number of synaptic boutons were used as parameters to assess the developmental status of the NMJ in the different genotypes. We determined the relationship between DNlg4 and DNrx in regulating the development of the NMJ by comparing the developmental defects in the different genotypes. Results Deletion of DNrx result ed in more severe developmental defects in NMJs than DNlg4 deletion, characterized by shorter synaptic branches and fewer boutons. Loss of DNlg4 further aggravated the NMJ defects caused by DNrx deletion. The expression of exogenous DNlg4 in motoneurons partially rescued the developmental defects of NMJs caused by loss of DNrx. No in vivo physical interaction between DNlg4 and DNrx was detected. Conclusions DNlg4 and DNrx have synergistic effects on the positive regulation of synaptic development in Drosophila larvae, and DNlg4 regulates synaptic development in a DNrx-independent manner.