The soluble N‐ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex comprises synaptosome‐associated protein of 25 kDa (SNAP25), syntaxin‐1a (syx‐1), and synaptobrevin 2, which is essential for many physiologic processes requiring membrane fusion. Several studies imply that the loop region of SNAP25 plays important roles in SNARE‐complex assembly. However, why and how the flexible loop facilitates the complex assembly remains poorly understood because it is purposely deleted in almost all structural studies. By using NMR spectroscopy and circular dichroism spectropolarimetry, we characterized SNAP25 structure and interactions with other SNAREs in aqueous buffer and in the membrane. We found that the N‐terminal of the SNAP25 loop region binds with membrane, and this interaction induced a disorder‐to‐order conformational change of the loop, resulting in enhanced interaction between the C‐terminal of the SNAP25 loop and syx‐1. We further proved that SNARE‐complex assembly efficiency decreased when we disrupted the electrostatic interaction between C‐terminal of the SNAP25 loop and syx‐1, suggesting that the SNAP25 loop region facilitates SNARE‐complex assembly through promoting prefusion SNARE binary complex formation. Our work elucidates the role of the flexible loop and the membrane environment in SNARE‐complex assembly at the residue level, which helps to understand membrane fusion, a fundamental transport and communication process in cells.—Jiang, X., Zhang, Z., Cheng, K., Wu, Q., Jiang, L., Pielak, G. J., Liu, M., Li, C. Membrane‐mediated disorder‐to‐order transition of SNAP25 flexible linker facilitates its interaction with syntaxin‐1 and SNARE‐complex assembly. FASEB J. 33, 7985–7994 (2019). http://www.fasebj.org