Alzheimer's disease (AD) is the most common form of neurodegenerative dementia among the elderly. Excitotoxicity has been implicated as playing a dominant role in AD, especially related to the hyperactivation of excitatory neurons. Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin-dependent kinase and involved in the pathogenesis of AD, but the roles and mechanisms of DAPK1 in excitotoxicity in AD are still uncertain.We mainly explored the underlying mechanisms of DAPK1 involved in the excitotoxicity of AD and its clinical relevance.Differentiated SH-SY5Y human neuroblastoma cells, PS1 V97 L transgenic mice, and human plasma samples were used. Protein expression was assayed by immunoblotting, and intracellular calcium and neuronal damage was analyzed by flow cytometry. Plasma DAPK1 was measured by ELISA.We found that DAPK1 was activated after amyloid-β oligomers (AβOs) exposure in differentiated SH-SY5Y cells. Besides, we found the phosphorylation of GluNR2B subunit at Ser1303 was increased, which contributing to excitotoxicity and Ca2 + overload in SH-SY5Y cells. Inhibiting DAPK1 activity, knockdown of DAPK1 expression, and antagonizing GluN2B subunits could effectively prevent AβOs-induced activation of GluN2B subunit, Ca2 + overload, and neuronal apoptosis. Additionally, we found that DAPK1 was elevated in the brain of AD transgenic mouse and in the plasma of AD patients.Our finding will help to understand the mechanism of DAPK1 in the excitotoxicity in AD and provide a reference for the diagnosis and therapy of AD.