Colorectal cancer (CRC) is a typical lifestyle-related disease, and it metastasizes mostly to the liver. It is important to understand the molecular mechanisms of CRC metastasis in order to design new and effective treatments for CRC patients. Chemokines are known to have antitumor effects as their chemoattractant properties stimulate the accumulation of infiltrating immune cells (TILs) in tumors. Chemokine (C-X-C motif) ligand 16 (CXCL16), also known as SR-PSOX, is a unique membrane-bound chemokine that induces the expression of its specific receptor CXCR6. We previously reported that the expression of CXCL16 by cancer cells enhances the recruitment of TILs, thereby improving the prognosis of CRC. It has since been reported that CXCL16/CXCR6 expression is involved in the metastasis of various types of cancer. However, there is no report of the association between CXCL16 expression and liver metastasis in CRC. In this study, we investigated the role of cancer-derived CXCL16 and the possibility of gene therapy using CXCL16. Therefore, we examined the metastasis of colon 38 SL4 cells to the liver in an experimental model. Following injection of cancer cells into the intraportal vein, CXCL16-expressing CRC cells drastically inhibited liver metastasis. We also found that CD8 T cells and natural killer T (NKT) cells, known as CXCR6-expressing cells, increased in CXCL16-expressing metastatic tissue. Collectively, the inhibitory effect on metastasis to the liver by CXCL16 was observed in NKT cell-depleted mice but not in CD8 T cell-depleted mice. These results demonstrate the inhibitory effect of CXCL16 on liver metastasis via NKT cells in CRC.