The ribonuclear protein TDP-43 has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), with genetic mutations being linked to the neurological symptoms of the disease. Though alterations in the intracellular distribution of TDP-43 have been observed in skeletal muscles of patients suffering from ALS, it is not clear whether such modifications play an active role in the disease or merely represent an expression of muscle homeostatic mechanisms. Also, the molecular and metabolic pathways regulated by TDP-43 in the skeletal muscle remain largely unknown. Here, we analyze the function of TBPH, the Drosophila melanogaster ortholog of TDP-43, in skeletal muscles. We modulated the activity of TDP-43 in Drosophila muscles by means of RNA interference and observed that it is required to promote the formation and growth of neuromuscular synapses. TDP-43 regulated the expression levels of Disc-large (Dlg), and restoring Dlg expression either in skeletal muscles or in motoneurons was sufficient to suppress the locomotive and synaptic defects of TDP-43-null flies. These results were validated by the observation of a decrease in Dlg levels in human neuroblastoma cells and iPSC-differentiated motoneurons derived from ALS patients, suggesting similar mechanisms may potentially be involved in the pathophysiology of the disease. Our results help to unveil the physiological role of TDP-43 in skeletal muscles as well as the mechanisms responsible for the autonomous and non-autonomous behavior of this protein concerning the organization of neuromuscular synapses.