Siglec‐7 is a sialic acid binding receptor with inhibitory potential, expressed on human NK cells and monocytes. It has an unusual binding preference for α2,8‐linked disialic acids, such as those displayed by ganglioside GD3. Here we have investigated whether siglec‐7‐GD3 interactions are able to modulate NK cell cytotoxicity. Using synthetic polyacrylamide glycoprobes, siglec‐7 was found to be masked at the NK cell surface but it could be unmasked by sialidase treatment of NK cells. GD3 synthase‐transfected P815 target cells expressed high levels of GD3 and bound strongly to recombinant siglec‐7‐Fc protein. Surprisingly, GD3 synthase‐transfected P815 cells were killed more effectively by untreated cells in a siglec‐7‐independent manner. However, following sialidase treatment of NK cells, a siglec‐7‐dependent inhibition of killing was observed. These findings have important implications for NK cell cytotoxicity against tumor cells like melanoma that express high levels of GD3 ganglioside.