Retrograde axonal transport of recombinant adenoviral vectors has been used successfully to deliver genes to motoneurons in rodents after injection of the vectors into muscles. However, only a small proportion of motoneurons take up and retrogradely transport adenoviral particles, limiting the value of this gene delivery method for the treatment of motor neuron diseases (MNDs). Here we validate a new pharmacological approach for enhancing motoneuronal gene transfer after intramuscular injection of recombinant adenoviruses. We injected botulinum neurotoxin A (BoNT) into muscles of normal C57BL/6 mice and transgenic mice expressing the G93A mutation in the superoxide dismutase 1 gene (SOD1-G93A mutation, a model of amyotrophic lateral sclerosis) several days before inoculation with adenoviruses. Treatment with BoNT significantly enhanced gene transfer to motoneurons innervating the injected muscles. Modifications in motoneuron transduction appear to be a consequence of toxin-induced nerve sprouting at the end plates. These findings have major implications for devising protocols for preclinical and clinical studies using intramuscular injection of retrogradely transported gene vectors.