Lung cancer is the leading cause of cancer death worldwide. The spectrum of aberrations affecting signalling pathways in lung cancer pathogenesis has not been fully elucidated. Physiological expression of Vav1 is restricted to the haematopoietic system, where its best-known function is as a GDP/GTP nucleotide exchange factor for Rho/RacGTPases, an activity strictly controlled by tyrosine phosphorylation downstream of cell surface receptors. Here we find Vav1 expression in 42% of 78 lung cancer cell lines analysed. Moreover, immunohistochemical analysis of primary human lung cancer tissue samples revealed Vav1 expression in 26/59 malignant samples, including adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. Stronger Vav1 staining was associated with larger tumour size. siRNA-mediated knockdown of Vav1 in lung cancer cells reduced proliferation in agar and tumour growth in nude mice, while control siRNA had no effect, suggesting that Vav1 plays a critical role in the tumorigenicity of lung cancer cells. Vav1 is tyrosine-phosphorylated in lung cancer cells following activation by the growth factors EGF and TGFalpha, suggesting its participation in signalling events in these cells. Depletion of Vav1 reduced Rac-GTP activation and decreased expression of TGFalpha, an autocrine growth factor. These data suggest that Vav1 plays a role in the neoplastic process in lung cancer, identifying it as a potential therapeutic target for lung cancer therapy.