We previously reported that differentiation-inducing factor-1 (DIF-1) inhibited lung colony formation in mouse models of cancer metastasis by suppressing cancer cell proliferation and motility. Although adhesion of circulating tumor cells to vascular endothelial cells is an essential process for the initiation of tumor metastasis formation, the effects of DIF-1 on this process has not been elucidated. In this study, therefore, we investigated the effect of DIF-1 on the adhesion ability of tumor cells (human melanoma cells A2058, mouse melanoma cells B16BL6 and human colon cancer cells HCT116) to human umbilical vein endothelial cells (HUVECs). Treatment with DIF-1 significantly inhibited these cancer cells adhesion to HUVECs. To elucidate the mechanism of DIF-1 action, we analyzed adhesion-related genes in HUVECs. We found that the expression of vascular endothelial cell adhesion molecule-1 (VCAM-1) was suppressed by DIF-1 and the protein synthesis inhibitor cycloheximide abolished this inhibitory effect. We further evaluated the effect of DIF-1 using the lung metastasis model. Surprisingly, only 3 days administration of DIF-1 prior to inoculation of B16BL6 melanoma cells significantly inhibited lung colony formation. These results suggest that the main mechanism of DIF-1 anti-metastatic effect might be attenuation of adhesion between circulating tumor cells to blood vessels.