To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca²⁺ and metabolism, and Ca²⁺ efficacy of the exocytotic system in rat pancreatic islets. Fifteen hours exposure to 5 mM D--hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca²⁺-independent and ATP-independent component of insulin release, which was suppressed by GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was not affected 10 min after exposure to high glucose. Additionally, [Ca²⁺]_i efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP/ADP ratio in the HB-cultured islets in the presence of high glucose were lower, while glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB and that the decrease in ATP production is due to decreased NADH content in the mitochondria matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to D-HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.