Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl--fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic β cells. In ClC-3-/- mice, insulin secretion evoked by membrane depolarization (high extracellular K+, sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a ∼80% reduction in depolarization-evoked β cell exocytosis (monitored as increases in cell capacitance) in single ClC-3-/- β cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl- fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process. © 2009 Elsevier Inc. All rights reserved.