Non–small cell lung cancer is characterized by a specific metastatic pattern. The mechanism for organ-specific metastasis is poorly understood, although 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 non–small cell lung cancer metastases. Our results indicate that both non–small cell lung cancer tumor specimens resected from patients and non–small cell lung cancer cell lines express CXCR4, but not CXCL12. Non–small cell lung cancer cell lines undergo chemotaxis in response to CXCL12. CXCL12–CXCR4 activation of non–small cell lung cancer cell lines showed intracellular calcium mobilization and mitogen-activated protein kinase activation with enhanced extracellular signal-related kinase-1/2 phosphorylation without change in either proliferation or apoptosis. Target organs in a murine model that are the preferred destination of human non–small cell lung cancer metastases elaborate higher levels of CXCL12 than does the primary tumor, and suggest the generation of chemotactic gradients. The administration of specific neutralizing anti-CXCL12 antibodies to severe combined immunodeficient mice expressing human non–small cell lung cancer abrogated organ metastases, without affecting primary tumor-derived angiogenesis. These data suggest that the CXCL12–CXCR4 biological axis is involved in regulating the metastasis of non–small cell lung cancer.