As a SNARE binding protein, tomosyn has been reported to negatively regulate synaptic exocytosis via arresting syntaxin-1 and SNAP-25 into a nonfusogenic product that precludes synaptobrevin-2 entry, raising the question how the assembly of the SNARE complex is achieved. Here, we have investigated new functions of tomosyn in SNARE complex formation and SNARE-mediated vesicle fusion. Assisted by NSF/α-SNAP, syntaxin-1 escapes tomosyn arrest and assembles into the Munc18-1/syntaxin-1 complex. Munc13-1 then catalyzes the transit of syntaxin-1 from the Munc18-1/syntaxin-1 complex to the SNARE complex in a manner specific to synaptobrevin-2 but resistant to tomosyn. Our data suggest that tomosyn ensures SNARE assembly in a way amenable to tight regulation by Munc18-1 and Munc13-1.