Mannose-binding lectin (MBL) is a pattern recognition protein that plays an important role in innate immunity capable of activating the lectin pathway of the complement system. In this study, a MBL homologue (OnMBL) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnMBL is 687 bp of nucleotide sequence encoding polypeptides of 228 amino acids. The deduced amino acid sequence is highly homology to teleost and similar to mammalian MBL, containing a canonical collagen-like region, a carbohydrate recognition domain and a neck region. Expression analysis revealed that the OnMBL was highly expressed in the liver, and also exhibited in other tissues including hind kidney, intestines, head kidney and spleen. In addition, the OnMBL expression was significantly up-regulated in spleen and head kidney following challenges with a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). Recombinant OnMBL ((r)OnMBL) protein was able to agglutinate both S. agalactiae and A. Hydrophila in vitro. Taken together, the results of this study indicated that OnMBL, possessing apparent agglutination ability to bacterial pathogens, might be involved in host defense against bacterial infection in Nile tilapia.