Mast cells orchestrate the allergic response through the release of proinflammatory mediators, which is driven by the fusion of cytoplasmic secretory granules with the plasma membrane. During this process, SNARE proteins including Syntaxin4, SNAP23 and VAMP8 play a key role. Following stimulation, the kinase IKKβ interacts with and phosphorylates the t-SNARE SNAP23. Phosphorylated SNAP23 then associates with Syntaxin4 and the v-SNARE VAMP8 to form a ternary SNARE complex, which drives membrane fusion and mediator release. Interestingly, mast cell degranulation is impaired following exposure to bacteria such as Escherichia coli. However, the molecular mechanism(s) by which this occurs is unknown. Here, we show that E. coli exposure rapidly and additively inhibits degranulation in the RBL-2H3 rat mast cell line. Following co-culture with E. coli, the interaction between IKKβ and SNAP23 is disrupted, resulting in the hypophosphorylation of SNAP23. Subsequent formation of the ternary SNARE complex between SNAP23, Syntaxin4 and VAMP8 is strongly reduced. Collectively, these results demonstrate that E. coli exposure inhibits the formation of VAMP8-containing exocytic SNARE complexes and thus the release of VAMP8-dependent granules by interfering with SNAP23 phosphorylation.