The discovery of Toll-like receptors led to new insights into cellular immune processes. These receptors, as well as their genetic polymorphisms, are attributed an important role in the development of many diseases, including cancer. Based on the available literature, this is the first research into the role of the rs2072493/N592S polymorphism in the TLR5 gene in lung cancer. The objectives of this work were a functional evaluation of the variant as well as the identification of the proliferative capacity of cells carrying the variant. Healthy lung and lung cancer cell lines with inserted plasmids or adenovirus constructs containing wild-type or the rs2072493/N592S variant were used in the research. Following the stimulation of the thus obtained cells with flagellin, the ability of signal transmission, specific protein expression, as well as cell viability and migration were measured in the cells. The N592S variant was found to be functional, and it was established that after stimulation with flagellin, the increase in NF-κB activity in cells with the variant was lower. On the protein level, differences in the activity of downstream signaling molecules depending on the presence of the variant were observed, as well as a significantly lower mobility and viability of cells with the variant in comparison to wild-type TLR5 gene.