Poor clinical outcome of lung cancer (LuCa) is primarily due to lack of knowledge about specific molecules involved in its progression and metastasis. In this study, we for the first time show the clinical and biological significance of CC chemokine receptor-9 (CCR9) in non-small cell lung cancer (NSCLC). Expression of CCR9 and CCL25, the only natural ligand of CCR9, was significantly higher (p<0.0001) in NSCLC tissues and serum respectively, compared to their respective controls. Interestingly, expression of both CCR9 and CCL25 was significantly higher in adenocarcinomas (ACs) compared to squamous cell carcinomas (SCCs) (p = 0.04, and p< 0.0001). Similar to tissues, AC and SCC cell lines were positive for CCR9 expression. Despite of marginal difference in CCR9 expression, AC cells showed higher migratory and invasive potential in response to CCL25, compared to SCC cells. This differential biological response of AC cells was primarily due to differential expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases under the influence of CCL25. Our results suggest CCR9 as a potential target for developing new treatment modality for NSCLC. Additionally, differential serum CCL25 level in ACs and SCCs, two NSCLC subtypes, suggest its potential as a non-invasive diagnostic/prognostic biomarker.