Compromised cellular energy metabolism, cerebral hypoperfusion, and neuronal calcium dysregulation are involved in the pathological process of Alzheimer's disease (AD). ATP-sensitive potassium (KATP) channels in plasma membrane and inner mitochondrial membrane play important roles in modulating neuronal excitability, cell survival, and cerebral vascular tone. To investigate the therapeutic potential of drugs that activate KATP channels in AD, we first characterized the effects of the KATP channel opener diazoxide on cultured neurons, and then determined its ability to modify the disease process in the 3xTgAD mouse model of AD. Plasma and mitochondrial membrane potentials, cell excitability, intracellular Ca2+ levels and bioenergetics were measured in cultured cerebral cortical neurons exposed to diazoxide. Diazoxide hyperpolarized neurons, reduced the frequency of action potentials, attenuated Ca2+ influx through NMDA receptor channels, and reduced oxidative stress. 3xTgAD mice treated with diazoxide for 8 months exhibited improved performance in a learning and memory test, reduced levels of anxiety, decreased accumulation of Aβ oligomers and hyperphosphorylated tau in the cortex and hippocampus, and increased cerebral blood flow. Our findings show that diazoxide can ameliorate molecular, cytopathological, and behavioral alterations in a mouse model of AD suggesting a therapeutic potential for drugs that activate KATP channels in the treatment of AD.