Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) is highly expressed in invasive lung cancers. In vitro investigation indicated that SCUBE3 may play a critical role in lung cancer invasion and metastasis. The current study immunohistochemically investigated the expression of SCUBE3 in 119 cases of non-small cell lung cancer (NSCLC) tumors and this study evaluated its clinical-pathological and prognostic significance. SCUBE3 was found to be up-regulated in NSCLC tissue samples compared to adjacent normal tissue. High SCUBE3 expression was noted in 84/119 (70.6%) of NSCLC tissue samples and was positively correlated with lymph node involvement (p = 0.001) and advanced stages of tumor/lymph node metastasis (TNM) (p = 0.014). Furthermore, high SCUBE3 expression was significantly associated with loss of the epithelial marker E-cadherin (p = 0.0015) and acquisition of expression of the mesenchymal marker vimentin (p = 0.005). Patients with high SCUBE3 expression had significantly a shorter survival time compared to patients with low SCUBE3 expression (p = 0.001), and SCUBE3 expression served as an independent prognostic factor for NSCLC patients. Results indicated that SCUBE3 might be involved in regulating the epithelial-mesenchymal transition (EMT) and malignant progression in NSCLC. Results also indicated that SCUBE3d may be a potential therapeutic target for lung cancers.