It has been demonstrated that cancer cells that have survived cancer treatment may be more malignant than the original cancer cells. These cells are considered to be the main cause of metastasis in prognosis. A Nanog-overexpressing colon-26 (Nanog+colon26) was generated and its metastatic potential was confirmed to be enhanced, indicating higher malignancy. Extracellular vesicles (EVs) secreted from the cell line (Nanog+colon26EVs) were administered to mice at 5 μg nine times (three times per week for three weeks) via the tail vein (PBS was used as a control). Subsequently, Nanog+colon26 cells were administered to mice via the spleen, and the quantity of metastatic colonies in the liver was analyzed two weeks later. The results demonstrated that the administration of EVs suppressed metastasis. The enhanced phagocytic activity of macrophages observed in the group treated with Nanog+colon26EVs indicated the involvement of the immune system in the observed metastasis-suppressing effect. Small RNA sequencing was conducted to identify Nanog-dependent miRNAs that exhibited significant changes (Fc>=1.5 or Fc