The CXCR4 chemokine receptor plays an essential role in the homing of cells to organs expressing its ligand, CXCL12. CXCR4 expressed on tumor cells might regulate their traffic during metastasis. Here, we investigated whether the activation of CXCR4 on B16 murine melanoma cells regulates biological functions associated with metastasis, in vitro and in vivo. Flow cytometry and PCR analysis showed that B16 constitutively expresses high levels of CXCR4 (CXCR4-B16). Biological assays showed that the activation of CXCR4, by its ligand CXCL12, increases the migration, invasion, and proliferation of CXCR4-B16. AMD3100 significantly inhibited the stimulatory migrating effect induced by CXCL12. Treatment of CXCR4-B16 with CXCL12 increases their adhesion to liver sinusoidal endothelial cell (LSEC) monolayers. LSEC, expressing CXCL12, increased the migration of CXCR4-B16. In a liver metastasis model, CXCR4-B16 metastasis was associated with an increased expression of CXCL12 in LSEC territories. CXCR4-B16 cells were located close to LSEC microenvironments expressing CXCL12. Increased liver metastasis was observed after injecting CXCR4-B16 cells previously treated with CXCL12. Our results provide evidence showing that CXCR4 plays an important role in regulating biological functions associated with B16 liver metastasis.