Toll-like receptor 5 (TLR5), which is highly conserved from lower to higher vertebrates, is an important pattern recognition receptor (PRR) for bacterial flagellin. However, a soluble form of TLR5 (TLR5S) was identified in fish that is not present in mammals. To better understand the transcriptional regulation of TLR5S gene in fish, we determined the TLR5S 5′-flanking sequence region from flounder (Paralichthys olivaceus) and assayed its promoter activity in Hirame natural embryo (HINAE) cells. The 5′-flanking region of TLR5S (715 bp) contains sequence elements for two AP-1 binding sites, two C/EBP sites, and one NF-κB site. To elucidate the functional significance of these sites, deletion clones and a site-directed mutant of NF-κB were generated. We estimated the luciferase activity in flagellin- or lipopolysaccharide-stimulated HINAE cells. The co-transfection of p65 with the wild-type TLR5S promoter greatly increased luciferase activity by more than nine-fold compared with the NF-κB mutant. Wild-type TLR5S promoter activity was increased synergistically by more than 159.5-fold in the presence of flagellin and p65. Furthermore, it was determined that the level of TRL5S mRNA was up-regulated by p65 and flagellin using a quantitative PCR. Additionally, translocation of TLR5S in the HINAE-TLR5S stable cell line after flagellin stimulation was observed by confocal microscopy. These results suggest that NF-κB and flagellin are essential components that act as a transcription factor and ligand, respectively, for maximal induction of the TLR5S promoter.