The mechanism by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.