Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, is a critical regulator of various cancers. NLK expression was evaluated by Western blot in 8 paired fresh non-small-cell lung cancer (NSCLC) tissues and immunohistochemistry (IHC) on 83 paraffin-embedded slices. NLK was lowly expressed in NSCLC and significantly associated with NSCLC histological differentiation, clinical stage, lymph node status, and Ki-67. Multivariate analysis indicated that low NLK expression was an independent prognostic factor for NSCLC patients' low survival rate. In vitro, after the release of NSCLC cell line A549 from serum starvation, the expression of NLK was downregulated, whereas the cell-cycle-related proteins were upregulated. In addition, we used RNA interference to knock down NLK expression, then observed its effects on NSCLC's growth in vitro. Western blot analyses indicated that deletion of NLK was positively correlated with cell-cycle-related proteins. The present investigation demonstrated that suppression of NLK expression resulted in significant promotion of proliferation in NSCLC cells. And flow cytometry further indicated that loss of NLK promoted cell proliferation by facilitating S-phase and mitotic entry. Besides, the transcription activity of β-catenin/TCF in A549 cells was remarkably enhanced when NLK was knocked down, which suggested that NLK participated in NSCLC cell proliferation via medulating Wnt signaling pathway. Based on these findings, we can provide a potential strategy for NSCLC therapy. J. Cell. Biochem. 115: 81–92, 2014. © 2013 Wiley Periodicals, Inc.