Intracellular membrane traffic involves controlled membrane fission and fusion and is essential for eukaryotic cell homeostasis. Most intracellular fusion is facilitated by Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, which catalyze membrane merging by assembly of a coiled helical bundle of four 60- to 70-residue "SNARE motifs." Perhaps no intracellular fusion reaction is as tightly regulated as that at the neuronal synapse, mediated by the synaptic vesicle SNARE Synaptobrevin-2 and the presynaptic plasma membrane SNAREs Syntaxin-1a and SNAP25. SNAP25 is different from its partner SNAREs: it contributes not one but two SNARE motifs to the final complex and instead of transmembrane domains is anchored in the membrane by post-translational palmitoylation of a long flexible linker between the SNARE motifs. Despite reports of structural and functional differences between the two SNARE motifs, many models of SNARE assembly and fusion consider SNAP25 to be a single functional unit and do not address how linking two distinct motifs in a single polypeptide contributes to synaptic SNARE assembly and fusion. To investigate whether SNAP25's two SNARE motifs regulate each other's folding and ability to assemble with other SNAREs, we determined their secondary structures in isolation and in the context of the whole protein by NMR spectroscopy and correlated the ability of the individual membrane-anchored SNARE motifs to interact with Syntaxin-1a and catalyze fusion in FRET-based binding and single-particle fusion assays, respectively. Our results demonstrate that the isolated N-terminal SNARE motif of SNAP25 promotes stronger Syntaxin-1a binding on membranes and more efficient fusion than wild-type SNAP25, while the C-terminal SNARE motif binds only transiently and facilitates kinetically delayed fusion. By comparing the functional properties of the single motifs to those of the full-length protein, we propose a new model of SNAP25 self-regulation in SNARE assembly and membrane fusion.