Highly metastatic and chemotherapy-resistant properties of malignant melanomas stand as challenging barriers to successful treatment; yet, the mechanisms responsible for their aggressive characteristics are not fully defined. We show that a distinct population expressing CD133 (Prominin-1), which is highly enriched after administration of a chemotherapeutic drug, dacarbazine, has enhanced metastatic potential in vivo. CD133⁺ tumor cells are located close to tumor-associated lymphatic vessels in metastatic organs such as the regional lymph nodes and lung. Lymphatic endothelial cells promote the migratory activity of a CD133⁺ subset to target organs and regulation of lymphatic growth efficiently modulates the metastasis of CD133⁺ tumor cells. We found that lymphatic vessels in metastatic tissues stimulate chemokine receptor 4 (CXCR4)⁺/CD133⁺ cell metastasis to target organs by secretion of stromal cell–derived factor-1 (SDF-1). The CXCR4⁺/CD133⁺ cells exhibited higher metastatic activity compared with CXCR4⁻/CD133⁺ cells and, importantly, blockade of CXCR4 coupled with dacarbazine efficiently inhibited both tumor growth and metastasis; dacarbazine alone could not attenuate tumor metastasis. The current study demonstrates a previously unidentified role of the lymphatic microenvironment in facilitating metastasis of chemoresistant melanoma cells via a specific chemotactic axis, SDF-1/CXCR4. Our findings suggest that targeting the SDF-1/CXCR4 axis in addition to dacarbazine treatment could therapeutically block chemoresistant CD133⁺ cell metastasis toward a lymphatic metastatic niche. Cancer Res; 70(24); 10411–21. ©2010 AACR.