Significance Every vesicle fusion reaction is driven by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins that zipper into tight complexes between membranes. Members of the Sec1/Munc18 (SM) family tightly regulate this process through interactions with a member of the syntaxin SNARE subfamily. However, seemingly different interaction modes have been found for different SM proteins and syntaxins. Here we show that the ER–Golgi SM protein Sly1 interacts not only with the N-peptide of the syntaxin Sed5, as previously thought, but also with its remainder. This corroborates the idea that SM proteins and syntaxins generally make use of two spatially separated binding sites. Remarkably, binding of Sly1 relaxes the autoinhibitory closed conformation of Sed5 and thereby accelerates SNARE complex formation.