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• A novel C-type lectin with a EPN/WND motif (OmCTL) was identified and characterized from Onychostoma macrolepis. • The expression levels of OmCTL gene were significantly upregulated after pathogen challenge. • Recombinant OmCTL can agglutinates various bacteria in vitro in the presence of Ca2+. • Recombinant OmCTL could bind some saccharides such as d-mannose, LPS and PGN and so on. C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila , suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila , in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis. [ABSTRACT FROM AUTHOR]