We reported that differentiation-inducing factor-1 (DIF-1) inhibited the proliferation in various cancer cells. In addition, DIF-1 prevented lung colony formation in a mouse model of metastatic melanoma by suppressing cell motility and proliferation. Although adhesion between circulating tumor cells and vascular epithelial cells is the essential process of tumor metastasis, DIF-1‘s effect on this process remains to be elucidated. In the present study, we investigated the anti-metastatic effects of DIF-1 using human A2058 melanoma cells, murine B16BL6 melanoma cells, human HCT116 colon cancer cells and human umbilical vein endothelial cells (HUVECs). In the in-vitro experiments, cancer cells labeled with an enhanced green fluorescent protein (eGFP) were treated with DIF-1 (30 µM) and cocultured with confluent HUVECs for 6 h. DIF-1 significantly inhibited cancer cell adhesion to HUVECs. To clarify the mechanism of DIF-1‘s action, adhesion-related genes were analyzed using flow cytometry, immunofluorescence and western blotting. Cell adhesion molecules on HUVECs such as intercellular cell adhesion molecule-1 (ICAM- 1), vascular endothelial cell adhesion molecule-1 (VCAM-1) and E-selectin were suppressed by DIF-1 treatment. Furthermore, b1 integrin and focal adhesion makers such as FAK, c-Src and Paxillin were decreased by DIF-1 treatment. In the in-vivo experiments, 8-week-old C57BL/6 mice were divided into groups treated with or without DIF-1, which was intragastrically administered 3 days (300 mg/kg/day) before inoculation of B16BL6 melanoma cells via teil vein. Mice were euthanized 14 days after tumor cell inoculation, and lungs were excised for analysis. DIF-1 significantly inhibited the lung colony formation quantified by counting the colony number and evaluating the expressions of GFP and melanoma-specific markers. These results suggested the possibility that DIF-1 exerts anti-metastatic effect through suppressing circulating cancer cell adhesion to blood vessels in distant metastatic site.