Pancreatic β cells secrete insulin by Ca²⁺-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in β cells to regulate Ca²⁺ influx for insulin secretion. β cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca²⁺ channel (VDCC) current density. In situ Ca²⁺ imaging of β cells within islets expressing a membrane-bound G-CaMP8b Ca²⁺ sensor demonstrated initial local Ca²⁺ signals at the ELKS-localized vascular side of the β cell plasma membrane, which were markedly decreased in ELKS-KO β cells. Mechanistically, ELKS directly interacted with the VDCC-β subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the β cell plasma membrane for polarized Ca²⁺ influx and first-phase insulin secretion from pancreatic islets.