Here we propose the use of adeno-associated virus (AAV) vectors as a non-invasive vehicle for the nervous system to deliver genes to spinal motoneurons, based on their retrograde transport from muscle. Long-term protein expression in lower cervical motoneurons was achieved after injections of AAV into the triceps, independently of serotypes 1, 2, or 5. Muscle injections of AAV5-neurotrophin 3 (NT3) resulted in a significant increase in the levels of NT3 in the cervical enlargement, compared to those obtained after injections of AAV5-GFP. Following a dorsal lesion at C4/C5, animals injected with AAV5-NT3 made fewer errors (footslips) in the horizontal ladder test compared to those injected with AAV5-GFP. In parallel, the number and length of corticospinal tract (CST) fibers circumventing the injury site were significantly increased in rats injected with AAV5-NT3. Compared to controls, we observed less astrogliosis and less CST axonal retraction and/or enhanced sprouting in animals injected with AAV5-NT3. In sum, we demonstrate here that the delivery of nt3 via retrograde transport of AAV from triceps to cervical motoneurons leads to reduced functional loss and anatomical reorganization of the CST following injury, without introducing additional injury to the spinal cord.