Stem cell-based therapy holds great potential for substituting degenerated motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Missing protocols for advanced differentiation of transplanted cells into MNs, immune rejection, and the lack of suitable ALS models for preclinical trials have slowed the development of effective therapies. Here, we employed multiplex genetic-editing to generate a novel human pluripotent stem cell line containing doxycycline (Dox)-inducible MNs-specific transcription factors and comprehensively modified immunomodulatory genes. We transplanted these cells into the spinal cord of ALS large animal models (SOD1G93A pigs and TIA1P362L rabbits), which faithfully recapitulate pathologies and symptoms observed in ALS patients. The transplanted cells could efficiently differentiate into functional MNs upon Dox treatment in vivo, distribute throughout the spinal cord and motor cortex via extensive migration, survive long-term without the need for immunosuppression. Notably, these MNs integrated into host neural circuits, as evidenced by their long projection of peripheral axons to target muscle and reformation of neuromuscular junctions. As result, pathologies and motor deficits were substantially ameliorated in both animal models.