Amyotrophic lateral sclerosis (ALS) is categorized into ~10% familial and ~90% sporadic cases. While familial ALS is caused by mutations in many genes of diverse functions, the underlying pathogenic mechanisms of ALS, especially in sporadic ALS (sALS), are largely unknown. Notably, about half of the cases with sALS showed defects in mitochondrial respiratory complex IV (CIV). To determine the causal role of this defect in ALS, we used transcription activator-like effector-based mitochondrial genome editing to introduce mutations in CIV subunits in rat neurons. Our results demonstrate that neuronal CIV deficiency is sufficient to cause a number of ALS-like phenotypes, including cytosolic TAR DNA-binding protein 43 redistribution, selective motor neuron loss and paralysis. These results highlight CIV deficiency as a potential cause of sALS and shed light on the specific vulnerability of motor neurons, marking an important advance in understanding and therapeutic development of sALS.