Significance Macroautophagy is essential for the maintenance of cellular homeostasis and physiology in mammals, and relies on vesicle fusion between the autophagosome and the lysosome, forming the autolysosome to degrade unwanted cytosolic contents for recycling. The membrane fusion between the autophagosome and lysosome requires ATG8 family proteins and autophagy-related SNARE proteins including Syntaxin17, VAMP8, and SNAP29, but with poorly understood mechanisms. In this study, through systemic biochemical and structural characterizations, we reveal three different states of the key autophagosomal SNARE protein Syntaxin17 and provide mechanistic insights into the autoinhibited state of Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. Our findings are valuable for further understanding the functions of Syntaxin17 in the autophagosome–lysosome fusion process.