Secretagogin, a Ca2+-sensor protein, can trigger signal transduction cascades promoting vesicular exocytosis in an activity- and Ca2+-dependent manner. Even though a role for secretagogin in vesicle trafficking was hypothesized, it’s structural motifs that govern any such function remain unknown. In silico search of the secretagogin interactome was used to select consensus protein hits from open-label databases. X-ray crystallography was employed to map the interaction motif. Multiple immunostaining coupled to superresolution microscopy aided the localization of recombinant structural modules that had been overexpressed in insulinoma cells, with subcellular fractionation validating the cytochemical data. The effect of secretagogin modules on hormone release was monitored by ELISA in gain of function experiments in vitro. Besides synaptosomal associated protein 25 kDa (SNAP-25), syntaxin-4 was identified an alternative binding partner for secretagogin. A C-terminal hydrophobic groove was invariably responsible for interactive partner binding. Truncation mutants for secretagogin failed to traffic properly or led to loss-of-function vesicular exocytosis. In the pancreas, secretagogin, syntaxin-4 and SNAP-25 were found preferentially co-expressed in α-cells. Thus, syntaxin-4 overexpression in β-like INS1E cells increased insulin release. We have defined the exact structural interactions by which secretagogin binds to either SNAP-25 or syntaxin-4. Our structural biology data provide support for tissue- or cell-type-specific secretagogin action, including an effect on the rate and kinetics of hormone release. A hydrophobic groove in the C-terminal domain of secretagogin mediates protein-protein interactions. This groove could be used as a template to search for novel interactive partners in the many secretagogin-expressing neuroendocrine tissues.