Non-small cell lung cancer (NSCLC) is characterized by a specific metastatic pattern. The mechanism for organ-specific metastasis is poorly understood, although recent evidence has suggested that the chemokine stromal derived factor-1 (CXCL12) and its cognate receptor CXCR4 may regulate breast cancer metastasis. We hypothesized that the CXCL12/CXCR4 biological axis is important in mediating NSCLC metastases. Our results indicate that both NSCLC tumor specimens resected from patients and NSCLC cell lines express CXCR4, but not CXCL12. NSCLC cell lines undergo chemotaxis in response to CXCL12. CXCL12/CXCR4 activation of NSCLC cell lines showed intracellular calcium mobilization and mitogen-activated protein kinase activation with enhanced extracellular signal regulated kinase 1/2 phosphorylation without a change in either proliferation or apoptosis. Target organs in the murine model that are the preferred destination of human NSCLC metastases elaborate higher levels of CXCL12 than the primary tumor and suggest the generation of chemotactic gradients. The administration of specific neutralizing anti-CXCL12 antibodies to SCID mice expressing human NSCLC abrogated organ metastases without affecting primary tumor-derived angiogenesis. The data suggest that the CXCL12/CXCR4 biological axis is involved in regulating the metastasis of NSCLC.