Interaction of syntaxin 1 with the α_1D subunit of the voltage-gated L type Ca²⁺ channel was investigated in the pancreatic β cell. Coexpression of the enhanced green fluorescent protein-linked α_1D subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the α_1D subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the α_1D subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca²⁺ channel activity because there was a faster Ca²⁺ current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K⁺ or exposure to high Ca²⁺. Exchanging Ca²⁺ for Ba²⁺ abolished the effect of antisyntaxin 1 on both Ca²⁺ channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca²⁺-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure–function relationship between the α_1D subunit of the L type Ca²⁺ channel and the exocytotic machinery in the pancreatic β cell.