Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in mammals and play an important role in innate immunity. In the present study, a TLR5M gene was cloned from turbot, Scophthalmus maximus, and its immune responsive expression was subsequently studied in vivo. The Scophthalmus maximus (Sm)TLR5M gene is 4268 bp in length, consists of four exons and three introns and encodes a peptide of 892 amino acids (aa). The deduced protein possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 23 LRR motifs, a transmembrane (TM) domain and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped SmTLR5M with other teleost TLR5Ms. A number of binding sites for transcription factors involved in immune response regulation were predicted in the 5′-flanking region of SmTLR5M. Quantitative real-time PCR (qPCR) analysis demonstrated that SmTLR5M mRNA was expressed ubiquitously with higher levels in head kidney and spleen. Its expression following stimulation with flagellin and lipopolysaccharide (LPS) was further tested in gills, spleen, head kidney and muscle. The maximum increases of SmTLR5M transcript levels ranged from 1.3 to 6.8-fold and appeared at 3 h to 5 day post-injection depending on different organs and stimuli. These findings suggest that SmTLR5M may play an important role in immune responses to infections with bacterial pathogens.