Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease in which motor neurons are primarily affected. Mutations in the TARDBP gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 inclusions accumulate in degenerating motoneurons in most cases of ALS. However, the cellular and molecular bases of TDP-43 toxicity that initiate and promote degeneration of motoneuron have been incompletely understood to date. Here, we developed a transgenic zebrafish model for ALS using Gal4-UAS system, in which TDP-43 toxicity can be investigated in an intact spinal motoneuron in vivo. We showed that overexpression of the wild-type zebrafish TDP-43 caused reduced axonal outgrowth and arborization in the CaP primary motoneuron in larval zebrafish. Further, in vivo two-photon calcium imaging revealed that the CaP motoneuron overexpressing TDP-43 displayed reduced calcium transients both in the cell body and presynaptic terminals during fictive swimming, indicating a reduced excitability. Consistent with this observation, pan-motorneuronal induction of TDP-43 resulted in an impaired locomotor activity. We also confirmed that the induction of the human TDP-43 with a mutation found in familial ALS caused qualitatively similar abnormalities in CaP motoneurons. Thus, we propose that the TDP-43 transgenic zebrafish is useful to characterize early presymptomatic neuronal dysfunction caused by TDP-43 and to develop potential therapeutic approaches for mitigating TDP-43 toxicity in ALS.