AbstractNK cells eliminate cancer and virus‐infected cells through their cytolytic activity. The last step in NK‐cell cytotoxicity, resulting in exocytosis of granule content, requires fusion of lytic granules with the plasma membrane. Proteins from the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) family mediate membrane fusion events in the cell. Here, we show that NK cells express all members of the R‐SNARE subgroup. Two of these R‐SNARE proteins, VAMP4 and VAMP7, colocalize with lytic granules during cytotoxic interactions. However, only VAMP7 associates with perforin‐containing granules in nonactivated cells, indicating that the two VAMPs have different functions in exocytosis. Using both the tumor NK‐cell line YTS and the peripheral NK cells, we show that the disruption of expression of either VAMP4 or VAMP7 inhibits the release of lytic granules and severely impairs NK‐cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in IFN‐γ secretion in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a more general function in NK‐cell activity than VAMP4.