Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX1's closed conformation driven by its Habc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.