In renal cell carcinoma (RCC), the presence of higher gangliosides correlates with systematic metastasis. Disialosyl globopentaosylceramide (DSGb5) was identified previously as one of the major gangliosides from RCC tissues. Siglec-7 (sialic acid-binding Ig-like lectin-7), expressed on natural killer (NK) cells as an inhibitory receptor, has a striking preference for internally branched α2,6-linked disialic gangliosides such as DSGb5. To clarify the functional role of DSGb5 in RCC metastases, we have investigated whether DSGb5 expressed on RCC cells can modulate NK cell cytotoxicity in a Siglec-7-dependent manner. The binding activity of RCC cells to Siglec-7-Fc fusion protein was specifically inhibited by anti-DSGb5 monoclonal antibody and transfection of siRNA for ST6GalNAcVI (synthetase of DSGb5). These observations showed that Siglec-7-Fc fusion protein specifically bound to DSGb5 expressed on RCC cells. In contrast, the sialic acid-binding site of Siglec-7 on NK cells was masked by cis interactions with endogenous sialoconjugates at the cell surface, but it could be unmasked by sialidase treatment of the NK cells. Following sialidase treatment of NK cells, NK cell cytotoxicity against RCC cells with high DSGb5 expression was significantly decreased relative to cells with low DSGb5 expression. These findings indicate that such NK cell cytotoxicity against RCC cells could be inhibited by the interaction between Siglec-7 on effecter cells and DSGb5 on target cells. The results of the present study suggest that DSGb5 expressed on RCC cells can downregulate NK cell cytotoxicity in a DSGb5-Siglec-7-dependent manner and that RCC cells with DSGb5 create favorable circumstance for their own survival and metastases.