TLR5S cDNA was cloned using homologous cloning and rapid amplification of cDNA ends techniques to study the regulatory role of TLR5S in the innate immune response of half-smooth tongue sole, Cynoglossus semilaevis, (designated Cs.TLR5S). Three alternative splicing variants (Cs.TLR5S x1, Cs.TLR5S x2, and Cs.TLR5S x3) of the Cs.TLR5S cDNA sequence were found in C. semilaevis. The full-length CsTLR5S cDNA included a 308 bp 5''-untranslated region (UTR), a 1701 bp open reading frame, and 138 bp, 364 bp, and 637 bp 3''-UTRs, respectively. The cDNA encoded a polypeptide of 567 amino acids, with a molecular mass of 64.03 kD and an isoelectric point of 8.49. Multiple sequence alignment revealed that the Cs.TLR5S proteins are well conserved with a typical modular architecture and identical active sites throughout vertebrates, and shared the highest identity with Paralichthys olivaceus TLR5S (61%), suggesting a conserved function for TLR5S. A phylogenetic analysis indicated that Cs.TLR5S and homologous TLR5S sequences from teleosts were clustered into a clade, and Cs.TLR5S was separated from another clade with amphibians, mammals, and other vertebrates. A tissue expression profile analysis using the quantitative real-time polymerase chain reaction (qRT-PCR) showed that Cs.TLR5S mRNA was constitutively expressed in all tested tissues, with predominant expression in liver and the lowest expression in spleen. Alternative splicing of the 3''-UTR using qRT-PCR showed that Cs.TLR5S x3 was only expressed in liver, whereas Cs.TLR5S x1 was expressed in liver and intestine. In addition, Cs.TLR5S was expressed at different levels in liver, spleen, intestine, and head kidney after a Vibrio anguillarum challenge. These results suggest that expression of the C. semilaevis Cs.TLR5S variants are differentially regulated in different tissues and play important roles in the immune response against bacterial pathogens.