NAD(P)H oxidase-derived H₂O₂ was recently proposed to act, in several cells, as the signal mediating the activation of volume-sensitive Cl^- channels (VSAC) under a variety of pathophysiological conditions. The present study aims at investigating whether a similar situation prevails in b cells derived BRIN-BD11 cells exposed to hypotonicity, a condition previously demonstrated to increase insulin secretion. Exogenous H₂O₂ also stimulated insulin secretion with a threshold concentration close to 38 mM and a maximal response at about 100 mM. The inhibitors of volume-sensitive anion channels 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid decreased the secretory response to exogenous H₂O₂, as well as to hypotonicity. In patch clamp experiments, exogenous H₂O₂ was observed to induce cell membrane depolarization that was also inhibited by NPPB, as previously reported for hypotonicity. Exposure of the BRIN-BD11 cells to a hypotonic medium cause a detectable increase in intracellular reactive oxygen species (ROS) that was abolished by diphenylene iodinium chloride (DPI), a NAD(P)H oxidase inhibitor. DPI as well as other NAD(P)H oxidase inhibitors such as phenyl arsine oxide and plumbagin nearly totally inhibited the insulin release provoked by exposure of the BRIN-BD11 cells to an hypotonic medium. Measurements of cell volume under hypotonic conditions showed that DPI like NPPB completely blocked the cell volume regulatory decrease otherwise observed within 15-20 min. On the other hand exogenous H₂O₂ did not change cell volume. Taken together, these data suggest that NAD(P)H oxidase-derived H₂O₂ appears the key intracellular messenger leading to opening of VSAC, hence membrane depolarization and the insulin secretory response observed in BRIN-BD11 cells to extracellular hypotonicity.