AbstractMediator release from mast cells (MC) is a crucial step in allergic and non‐allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N‐ethyl‐maleimide‐sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N‐ethylmaleide sensitive factor attachment protein (SNAP)‐23, Syntaxin (STX)‐1B, STX‐2, STX‐3, STX‐4, and STX‐6 but not SNAP‐25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)‐3, VAMP‐7 and VAMP‐8 and, in contrast to previous reports about rodent MC, only low levels of VAMP‐2. Furthermore, VAMP‐7 and VAMP‐8 were found to translocate to the plasma membrane and interact with SNAP‐23 and STX‐4 upon activation. Inhibition of SNAP‐23, STX‐4, VAMP‐7 or VAMP‐8, but not VAMP‐2 or VAMP‐3, resulted in a markedly reduced high‐affinity IgE receptor‐mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP‐7 and VAMP‐8, but not VAMP‐2, are required for rapid degranulation.