Oral administration of hot-water extract of Spirulina, cyanobacterium Spirulina platensis, leads to augmentation of NK cytotoxicity in humans. Here, we applied to syngeneic tumor-implant mice (C57BL/6 versus B16 melanoma) Spirulina to elucidate the mechanism of raising antitumor NK activation. A B16D8 subcell line barely expressed MHC class I but about 50% expressed Rae-1, a ligand for NK activation receptor NKG2D. The Rae-1-positive population of implant B16 melanoma was effectively eliminated in the tumor mass progressed in mice. This antitumor activity was induced in parallel with IFN-gamma and abolished in mice by treatment with asialoGM-1 but not CD8beta Ab, suggesting the effector is NK cell. NK cell activation occurred in the spleen of wild-type mice medicated with Spirulina. This Spirulina-mediated enhanced NK activation was abrogated in MyD88 -/- mice but not in TICAM-1 -/- mice. The NK activating properties of Spirulina depending on MyD88 were confirmed with in vitro bone marrow-derived dendritic cells expressing TLR2/4. In D16D8 tumor challenge studies, the antitumor effect of Spirulina was abolished in MyD88 -/- mice. Hence, orally administered Spirulina enhances tumoricidal NK activation through the MyD88 pathway. Spirulina exerted a synergistic antitumor activity with BCG-cell wall skeleton, which is known to activate the MyD88 pathway via TLR2/4 with no NK enhancing activity. Spirulina and BCG-cell wall skeleton synergistically augmented IFN-gamma production and antitumor potential in the B16D8 versus C57BL/6 system. We infer from these results that NK activation by Spirulina has some advantage in combinational use with BCG-cell wall skeleton for developing adjuvant-based antitumor immunotherapy.