NAD(P)H oxidase (NOX)-derived H2O2 was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 cells. Exogenous H2O2 stimulated insulin secretion with a threshold concentration close to 38 mM and a maximal response at about 100 mM. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H2O2. In patch clamp experiments, exogenous H2O2 was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenylene iodinium chloride (DPI), a general NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H2O2. Taken together, these data suggest that NOX-derived H2O2 plays a key role in the insulin secretory response of BRIN-BD11 cells to extracellular hypotonicity.