The anti-metastatic activity of MD-Fraction extracted from the maitake mushroom (Grifola frondosa) was examined in an experimental murine model of lung metastasis. Intraperitoneal administration of MD-Fraction 2 d before tumor implantation significantly inhibited lung metastasis of colon-26 carcinoma and B16/BL6 melanoma cells. In this model, MD-Fraction enhanced IL-12 production from antigen presenting cells (APCs). MD-Fraction treatment activated NK cells and increased cytotoxicity against YAC-1 and colon-26 carcinoma cells. Furthermore, the depletion of NK cells with anti-asialo GM1 abolished the inhibitory effect of MD-Fraction on lung metastasis of colon-26 cells. Ex vivo, B16/BL6 cell adhesion to LPS-activated murine lung vascular endothelial cells was inhibited by MD-Fraction and anti-intercellular adhesion molecule (ICAM)-1 antibody. These results suggest that MD-Fraction inhibits tumor metastasis by activating NK cells and APCs, and by suppressing of ICAM-1 leading to the inhibition of tumor cell adhesion to vascular endothelial cells.