Background: Apolipoprotein E4 (apoE4) is a major genetic risk factor of Alzheimer's disease. Its C-terminal-truncated apoE4 (Delta 272-299) has neurotoxicity by affecting mitochondrial respiratory function. However, the molecular mechanism(s) underlying the action of apoE4 (Delta 272-299) in mitochondrial function remain poorly understood. Methods: The impact of neuronal apoE4 (Delta 272-299) expression on ER stress, mitochondrial-associated membrane (MAM) formation, GRP75, calcium transport and mitochondrial impairment was determined in vivo and in vitro. Furthermore, the importance of ER stress or GRP75 activity in the apoE4 (Delta 272-299)-promoted mitochondrial dysfunction in neuron was investigated. Results: Neuronal apoE4 (Delta 272-299) expression induced mitochondrial impairment by inducing ER stress and mitochondrial-associated membrane (MAM) formation in vivo and in vitro. Furthermore, apoE4 (Delta 272-299) expression promoted GRP75 expression, mitochondrial dysfunction and calcium transport into the mitochondria in neuron, which were significantly mitigated by treatment with PBA (an inhibitor of ER stress), MKT077 (a specific GRP75 inhibitor) or GRP75 silencing. Conclusions: ApoE4 (Delta 272-299) significantly impaired neuron mitochondrial function by triggering ER stress, up-regulating GRP75 expression to increase MAM formation, and mitochondrial calcium overload. Our findings may provide new insights into the neurotoxicity of apoE4 (Delta 272-299) against mitochondrial function and uncover new therapeutic targets for the intervention of Alzheimer's disease.