Recent evidence highlights long noncoding RNAs (lncRNA) as crucial regulators of cancer biology that contribute to essential cancer cell functions such as cell proliferation, apoptosis, and metastasis. In non–small cell lung cancer (NSCLC), several lncRNAs' expressions are misregulated and have been nominated as critical actors in NSCLC tumorigenesis. LncRNA ANRIL was first found to be required for the PRC2 recruitment to and silencing of p15^INK4B, the expression of which is induced by the ATM–E2F1 signaling pathway. Our previous study showed that ANRIL was significantly upregulated in gastric cancer, and it could promote cell proliferation and inhibit cell apoptosis by silencing of miR99a and miR449a transcription. However, its clinical significance and potential role in NSCLC is still not documented. In this study, we reported that ANRIL expression was increased in NSCLC tissues, and its expression level was significantly correlated with tumor–node–metastasis stages and tumor size. Moreover, patients with high levels of ANRIL expression had a relatively poor prognosis. In addition, taking advantage of loss-of-function experiments in NSCLC cells, we found that knockdown of ANRIL expression could impair cell proliferation and induce cell apoptosis both in vitro and vivo. Furthermore, we uncover that ANRIL could not repress p15 expression in PC9 cells, but through silencing of KLF2 and P21 transcription. Thus, we conclusively demonstrate that lncRNA ANRIL plays a key role in NSCLC development by associating its expression with survival in patients with NSCLC, providing novel insights on the function of lncRNA-driven tumorigenesis. Mol Cancer Ther; 14(1); 268–77. ©2014 AACR.