Lung cancer ranks first in both prevalence and mortality rates among all types of cancer. Metastasis is the main cause of treatment failure. Biomarkers are critical to early diagnosis and prediction and monitoring of progressive lesions. Several biomarkers have been identified for lung cancer but none have been routinely used clinically. The present study assessed the diagnostic and prognostic value of cyclase-associated protein 1 (CAP1) for lung cancer. CAP1 mRNA abundance and protein content were determined by real-time PCR and western blot analysis and/or immunostaining in biopsy specimens (24 neoplastic and 6 non-neoplastic) freshly collected at surgical lung resection, in 82 pathologically banked lung cancer specimens and in cultured non-invasive (95-C) and invasive (95-D) lung cancer cells. Multivariate regression analysis was performed to correlate immunoreactive CAP1 signal with cancer type and stage. In vitro cell migration was performed to determine the effect of RNA interference-mediated CAP1 gene silencing on invasiveness of 95-D cells. These analyses collectively demonstrated that: i) both CAP1 mRNA abundance and protein content were significantly higher in neoplastic compared to non-neoplastic specimens and in metastatic compared to non-metastatic specimens but not different between adenocarcinoma and squamous cell carcinoma; ii) immunoreactive CAP1 signal was significantly stronger in metastatic specimens and 95-D cells compared to non-metastatic specimens and 95-C cells; and iii) RNA interference-mediated CAP1 gene silencing adequately attenuated the invasive capacity of 95-D cells in vitro. These findings suggest that overexpression of CAP1 in lung cancer cells, particularly at the metastatic stage, may have significant clinical implications as a diagnostic/prognostic factor for lung cancer.