Delta-like 4 (DLL4) is a membrane-bound ligand, which belongs to the Notch signaling pathway and plays important roles in angiogenesis and vascular development. The expression of DLL4 in non-small cell lung cancer (NSCLC) remains unclear. Therefore, DLL4 expression was detected in clinical specimens using quantum dots (QDs)-immunohistochemistry (IHC) and lung cancer cell lines by quantitative real-time polymerase chain reaction. The protein levels of DLL4 were decreased in the tumor tissues of NSCLC patients and lung cancer cell lines. Kaplan-Meier analysis indicated that low expression of DLL4 predicted poor survival rate of NSCLC patients. A549 and A427 cells transfected with pCMV-DLL4 exhibited reduced cell proliferation, migration and invasion using MTT assay, wound healing assay and Transwell assay. These data indicate that DLL4 represents a new prognostic biomarker for NSCLC, and DLL4 overexpression inhibits cell proliferation and metastasis in vitro.