Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause-effect relationships between mSOD1 and mitochondriopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.