Significance An intriguing feature of the nervous system is its plasticity—the remarkable lifelong capacity to change and adapt in light of intrinsic and extrinsic stimuli. Among the many different adaptive mechanisms that occur within the nervous system, changes in neurotransmission form an important plasticity-bestowing mechanism in the reconfiguration of neuronal circuits. Here, we reveal that physical activity and spinal cord injury can switch the neurotransmitter phenotype of the fast axial motoneurons to coexpress glutamate. Furthermore, our study shows that the adult vertebrate spinal motoneurons corelease glutamate alongside ACh in neuromuscular junctions to regulate motor behaviors. Thus, our findings suggest that fast motoneuron glutamatergic respecification enables a motor function-enhancing mechanism in vertebrates.