We report a novel role for the CXC-chemokine, CXCL5, in the proliferation and invasion of head and neck squamous cell carcinoma (HNSCC). Previously, we reported transcriptional up-regulation of CXCL5 in metastatic cells. In this study, we provide biological validation of these findings and show that CXCL5 is intimately involved in tumor cell proliferation, migration, and invasion. Cells derived from a lymph node metastasis, but not from a synchronous primary tumor, secreted CXCL5 as judged by Western blotting of conditioned media. We used RNA interference to generate cell lines (shL5) in which CXCL5 expression was greatly reduced, and tested whether this modulated the cell phenotype. shL5 cells showed decreased proliferation compared with cells harboring nontargeting control sequences. In addition, we found that the ability of shL5 cells to migrate and invade in vitro through a basement membrane substitute was greatly impaired compared with control cells. Finally, whereas control cells were highly tumorigenic in nude mice, the tumorigenic potential in vivo of shL5 cells was found to be ablated. Taken together, these data suggest that CXCL5 production contributes to both enhanced proliferation and invasion of squamous cell carcinomas and that targeting of chemokine pathways may represent a potential therapeutic modality for these lesions.