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Background and objective It has been proven that toll-like receptor 5 (TLR5) plaied an important role in the development of tumor. In our previous study, we found that the expression of TLR5 was remarkably higher in non-small cell lung cancer (NSCLC) tissues than that in normal tissues, but the activation of TLR5 signaling pathway in NSCLC was still unknown. The aim of this study is to investigate the expression of TLR5 in different types of NSCLC cell lines, and analyze the activity of the signaling pathway after stimulated by its specific exogenous ligand flagellin. Methods The TLR5 protein was detected by immunofluorescence and Western blot in three kinds of NSCLC cell lines, and the TLR5 mRNA was detected by RT-PCR. Select the cell line of TLR5 highest expression as the research object, and select the suitable concentration of flagellin. NF-κB luciferase activity was detected to validate the TLR5 activation pathway through inhibitory signaling pathways by 0 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL TLR5 antibody. The chosen cell line was transfected by TLR5 shRNA plasmid, and p-IKBα, IKBα, p-ERK1/2, ERK1/2 and p-JNK of untrasfected and transfected cells were detected in the activity of TLR5 signaling pathway by Western blot at 0 min, 10 min, 30 min and 60 min, respectively. Results The expression of TLR5 was the highest in the lung adenocarcinoma cell line SPC-A-1 by immunofluorescence, mainly expressed on the cell membrane. NF-κB luciferase activity of SPC-A-1 cells was the highest, and the activity was increased in a dose-dependent manner. 0.1 μg/mL flagellin could significantly increase the NF-κB luciferase activity (P0.05). SPC-A-1 cells transfected TLR5-shRNA were also stimulated by flagellin (0.1 μg/mL). At 0 min, 10 min, 30 min and 60 min, p-IKBα and p-JNK proteins could not be detected, and the levels of IKBα and ERK1/2 had no significant changes (P>0.05), but the levels of p-ERK1/2 significantly increased as time went on (P