In higher vertebrates, there is only a membranal TLR5 (TLR5M), which is crucial for host defense against microbes via MyD88 signaling pathway. In teleost, both TLR5M and soluble TLR5 (TLR5S) are identified, whereas the antibacterial mechanism of TLR5S is largely unknown. In this study, we studied the immune antibacterial mechanism of Cynoglossus semilaevis TLR5S homologue (named CsTLR5S). CsTLR5S, a 71.1 kDa protein, consists of 649 amino acid residues and shares 41.7 %-57.8 % overall sequence identities with teleost TLR5S homologues. CsTLR5S contains a single extracellular domain (ECD) composed of 12 leucine-rich repeats. CsTLR5S expression was constitutively identified and upregulated by bacterial infection in tissues. In vitro recombinant CsTLR5S (rCsTLR5S) could interact with bacteria and tongue sole rTLR5M (rCsTLR5M). Furthermore, rCsTLR5S could bind to the membranal CsTLR5M of peripheral blood leukocytes (PBLs), which led to enhancing the activity and the antibacterial role of PBLs via Myd88-NF-κB pathway. In vivo rCsTLR5S could activate the Myd88-NF-κB pathway, facilitate the release of proinflammatory cytokines, and enhance the host antibacterial response against Vibrio harveyi. Moreover, the knockdown of CsTLR5M or the Myd88 inhibitor could significantly suppress the antibacterial effect of rCsTLR5S. Collectively, our findings added important insights into the TLR5S immune antibacterial property in a TLR5M-MyD88-dependent manner.