Syntaxin-1, known as a multi-domain protein, regulates vesicle fusion by forming SNARE complex with synaptobrevin-2 and SNAP-25. However, the role of syntaxin-1 in synapse formation remains uncovered. Here we demonstrated that the expression level of syntaxin-1 was highly associated with the process of synaptogenesis. The R151A and I155A mutations but not Habc or transmembrane domain truncations of syntaxin-1 impaired its facilitation in synapse formation. Our results suggested that syntaxin-1 accelerated synapse formation via activating synaptic vesicle release.