Pancreatic islets express superoxide-producing NADPH oxidase system but its role remains unknown. To address this, we studied the mechanisms of impaired insulin secretion induced by diphenyleneiodium (DPI), an NADPH-oxidase inhibitor. We investigated the effects of DPI on glucose- and nonfuel-stimulated insulin secretion, islet glucose metabolism, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) dynamics in rat islets and -cell line RINm5F cells. DPI did not affect insulin secretion at 3.3 mM glucose but totally suppressed insulin secretion stimulated by 16.7 mM glucose (%control: 9.2±1.2%, P <0.001). DPI also inhibited insulin release by high K⁺-induced membrane depolarization (%control: 36.0±5.3%, P <0.01) and protein kinase C activation (%control: 30.2±10.6% in the presence of extracellular Ca²⁺, P <0.01, %control: 42.0±4.7% in the absence of extracellular Ca²⁺, P <0.01). However, DPI had no effect on mastoparan-induced insulin secretion at 3.3 and 16.7 mM glucose under Ca²⁺-free conditions. DPI significantly suppressed islet glucose oxidation and ATP contents through its known inhibitory action on Complex I in mitochondrial respiratory chain. On the other hand, DPI altered [Ca²⁺]_i dynamics in response to high glucose and membrane depolarization, and DPI per se dose-dependently increased [Ca²⁺]_i. The DPI-induced [Ca²⁺]_i rise was associated with a transient increase in insulin secretion and was attenuated by removing extracellular Ca²⁺, L-type voltage-dependent Ca²⁺ channel blockers, mitochondrial inhibitors or adding 0.1 or 1.0 µM H₂O₂ exogenously. Our results showed that DPI impairment of insulin secretion involved altered Ca²⁺ signaling, suggesting that NADPH oxidase may modulate Ca²⁺ signaling in cells.