Although syntaxin 1 is generally thought to function as the primary target-N-ethylmaleimide-sensitive factor attachment protein receptor required for pancreatic beta cell insulin secretion, we have observed that overexpression of a dominant-interfering syntaxin 4 mutant (syntaxin 4/DeltaTM) attenuated glucose-stimulated insulin secretion in betaHC-9 cells. Furthermore, these cells express the selective syntaxin 4-binding protein Synip (syntaxin 4 interacting protein), and Synip was specifically co-immunoprecipitated with syntaxin 4 but not syntaxin 1. Overexpression of the full-length Synip protein (Synip/wild type) inhibited VAMP2 association with syntaxin 4 and decreased glucose-stimulated insulin secretion. This did not occur with a Synip mutant (Synip/ DeltaEF) that was incapable of binding syntaxin 4. Consistent with a functional role of syntaxin 4 in this process, expression of syntaxin 4/DeltaTM also inhibited glucose-stimulated insulin secretion. Furthermore, analysis of first and second phase insulin secretion demonstrated that syntaxin 4/DeltaTM mainly suppressed the second phase of insulin secretion. In contrast, overexpression of Synip resulted in an inhibition of both the first and second phase of glucose-stimulated insulin secretion. These data demonstrate that syntaxin 4 plays a functional role on insulin release and granule fusion in beta cells and that this process is regulated by the syntaxin 4-specific binding protein Synip.