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SNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress.