In addition to their cell autonomous activities, a number of homeoprotein transcription factors transfer between cells and regulate gene transcription, protein translation and chromatin organization in a non-cell autonomous way. ENGRAILED-1 homeoprotein has the latter properties and is expressed in spinal cord V1 interneurons that synapse on large α-motoneurons. Because several homeoproteins have neuroprotective effects in the central nervous system, we analyzed the motor phenotype of mice with reduced extracellular spinal cord ENGRAILED-1 or lacking one functional Engrailed-1 allele. These mice show progressive muscle weakness, abnormal spinal reflex and partial neuromuscular junction denervation. In the Engrailed-1 heterozygote, large α-motoneuron degeneration is first observed at 4.5 months of age and progresses to reach 50 per cent loss in 15.5-month-old mice. A single intrathecal injection of exogenous recombinant human ENGRAILED-1 at 3 months in the lumbar enlargement of the heterozygote results in the addressing of the protein to α-motoneurons. This fully restores endplate innervation, limb strength and extensor reflex for at least 3 months, and prevents the death of lumbar α-motoneurons. Bioinformatic analysis revealed potential Engrailed-1 target genes that can interact with genes implicated in familial forms of Amyotrophic Lateral Sclerosis, and that one mechanism through which it exerts its trophic activity on α-motoneurons may involve the regulation of autophagy. These results demonstrate a novel non-cell autonomous function of ENGRAILED-1 in the spinal cord that sustains motoneuron survival and function, and that a single injection of the transcription factor in the Engrailed-1 heterozygote prevents α-motoneuron degeneration and strength loss for several months.