Axon injury is a central determinant of irreversible neurological deficit and disease progression in patients with multiple sclerosis (MS). CD8⁺ lymphocytes (CTLs) within inflammatory demyelinated MS lesions correlate with acute axon injury and neurological deficits. The mechanisms of these correlations are unknown. We interrogated CTL-mediated axon injury using the transgenic OT-I antigen-specific CTL model system in conjunction with a chambered cortical neuron culture platform that permitted the isolated manipulation of axons independent of neuron cell bodies and glia. Interferon gamma upregulated, through a dose dependent mechanism, the axonal expression of functional major histocompatibility complex class I (MHC I) molecules competent to present immunologically-relevant antigens derived from endogenously expressed proteins. Antigen-specific CTLs formed cytotoxic immune synapses with and directly injured axons expressing antigen-loaded MHC I molecules. CTL-mediated axon injury was mechanistically dependent upon axonal MHC I antigen presentation, T cell receptor specificity and axoplasmic granzyme B activity. Despite extensive distal CTL-mediated axon injury, acute neuron cell body apoptosis was not observed. These findings present a novel model of immune-mediated axon injury and offer anti-axonal CTLs and granzyme B as targets for the therapeutic protection of axons and prevention of neurological deficits in MS patients.