Toll-like receptors (TLRs) play a critical role in the immune process acting as innate sensors of pathogens. Toll-like receptor 5 (TLR5) is especially relevant in those tissues maintaining close contact with microorganisms, not only because it prevents infections but also due to its involvement in the regulation of host-commensal interactions. Recent studies suggest that the occurrence of genetic polymorphisms may impair TLR function, consequently increasing or decreasing the individual susceptibility to infectious diseases. In this study, the promoter sequence of the porcine TLR5 gene was scanned with the aim of identifying mutations with potential effects on gene expression. Two Indel variations in the predicted promoter sequence and seven single-nucleotide polymorphisms were identified. The luciferase reporter gene assay indicated that one Indel, consisting in a 23-bp insertion at the -581 to -559 nucleotide position, creates an additional STAT binding site, and it is associated with an increase of the promoter activity. This finding suggests that genetic variation in the TLR5 promoter could alter the expression of the gene, and may be used as a molecular marker to define pathogen susceptibility or resistance patterns in pigs.