AbstractCalcium/calmodulin-dependent protein serine kinase (CASK) is a key player in vesicle transport and release in neurons. However, its precise role, particularly in nonneuronal systems, is incompletely understood. We report that CASK functions as an important regulator of insulin secretion. CASK depletion in mouse islets/β cells substantially reduces insulin secretion and vesicle docking/fusion. CASK forms a ternary complex with Mint1 and Munc18-1, and this event is regulated by glucose stimulation in β cells. The crystal structure of the CASK/Mint1 complex demonstrates that Mint1 exhibits a unique “whip”-like structure that wraps tightly around the CASK-CaMK domain, which contains dual hydrophobic interaction sites. When triggered by CASK binding, Mint1 modulates the assembly of the complex. Further investigation revealed that CASK-Mint1 binding is critical for ternary complex formation, thereby controlling Munc18-1 membrane localization and insulin secretion. Our work illustrates the distinctive molecular basis underlying CASK/Mint1/Munc18-1 complex formation and reveals the importance of the CASK-Mint1-Munc18 signaling axis in insulin secretion.