Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis (ALS). CD4 ⁺ T cells that regulate a protective immunity during the neurodegenerative process have received the most attention. CD8 ⁺ T cells are also observed in the spinal cord of patients and ALS mice although their contribution to the disease still remains elusive. Here, we found that activated CD8 ⁺ T lymphocytes infiltrate the central nervous system (CNS) of a mouse model of ALS at the symptomatic stage. Selective ablation of CD8 ⁺ T cells in mice expressing the ALS-associated superoxide dismutase-1 (SOD1) ^G93A mutant decreased spinal motoneuron loss. Using motoneuron-CD8 ⁺ T cell coculture systems, we found that mutant SOD1-expressing CD8 ⁺ T lymphocytes selectively kill motoneurons. This cytotoxicity activity requires the recognition of the peptide-MHC-I complex (where MHC-I represents major histocompatibility complex class I). Measurement of interaction strength by atomic force microscopy-based single-cell force spectroscopy demonstrated a specific MHC-I-dependent interaction between motoneuron and SOD1 ^G93A CD8 ⁺ T cells. Activated mutant SOD1 CD8 ⁺ T cells produce interferon-γ, which elicits the expression of the MHC-I complex in motoneurons and exerts their cytotoxic function through Fas and granzyme pathways. In addition, analysis of the clonal diversity of CD8 ⁺ T cells in the periphery and CNS of ALS mice identified an antigen-restricted repertoire of their T cell receptor in the CNS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS.