Laryngeal squamous cell carcinoma is the most common form of head and neck squamous cell carcinoma. Multiple approaches have been applied to treat this type of cancer; however, no significant improvement in survival rate has been achieved. In the present study, the role of nemo-like kinase (NLK) in human laryngeal carcinoma Hep-2 cells was investigated. NLK has been identified as an important regulator of cell growth, patterning and cell death in a variety of organisms. Lentivirus-mediated-shRNA was employed to silence endogenous NLK expression. Downregulation of the expression of NLK following lentivirus infection was confirmed using reverse transcription quantitative polymerase chain reaction and western blot analysis. The effects of NLK downregulation on Hep-2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively. Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels. Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells. Furthermore, the absence of NLK may lead to cancer cell death. Collectively, the results of the present study demonstrated that the lentivirus-mediated targeted disruption of NLK may be a promising therapeutic method for the treatment of laryngeal cancer.