Purpose: Cyclooxygenase-2 (COX-2), one isoform of cyclooxygenase proinflammatory enzymes, plays an important role in tumor development and progression. Researches of human cancers have revealed high expression levels of COX-2 in a variety of cancers including lung cancer. The mechanism of COX-2 in the pathogenesis of non-small cell lung cancer (NSCLC) cells is not well understood. Methods: We constructed a lentivirus vector mediated RNA interference (RNAi) targeting COX-2 for the treatment of human NSCLC cells. RNAi technology was used to knockdown the expression of COX-2 in NSCLC cell lines. The efficiency and specificity was validated by quantitative real-time PCR and western blotting. The cell growth and cell cycle were determined by MTT and flow cytometry assay, respectively. Cell cycle-regulated gene expression, including cyclin D1, p21 and survivin, whose expression was modulated by COX-2, was also examined. Results: LV-COX-2-silencing (si)RNA lentivirus vector was effective and its inhibitory effects on COX-2 mRNA and protein expression was efficient and specific. Gene knockdown of COX-2 by LV-COX-2-siRNA significantly inhibited the growth and induced cell cycle arrest of NSCLC cell lines. In addition, silence of COX-2 mediated by LV-COX-2-siRNA modulated the expression of cell cycle-regulated gene, up-regulating p21 and downregulating cyclin D1 and survivin. Conclusions: Our findings imply that COX-2 and its signaling pathway may provide a novel therapeutic target for the treatment of NSCLC.