Transferrin (TF), an iron-binding glycoprotein, plays an important role in host defense against pathogenic infection, which inhibits the growth and proliferation of pathogens, deprives iron from invading pathogens, and activates anti-microbial responses in macrophages. In this study, a TF homologue (OnTF) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression pattern against bacterial infection and capability binding bacterial pathogens. The open reading frame of OnTF is 2118 bp of nucleotide sequence encoding polypeptides of 705 amino acids. The deduced protein is highly homology to the other species, containing two conserved iron binding lobes: N-lobe and C-lobe. Expression analysis revealed that the OnTF was extremely highly expressed in liver tissue; however, much weakly exhibited in other examined tissues including spleen and head kidney. The OnTF expression was significantly up-regulated in the liver, spleen and head kidney following infection of a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). The up-regulation of OnTF expression was also demonstrated in hepatocytes and macrophages in vitro stimulated with S. agalactiae and A. hydrophila. In addition, recombinant OnTF ((r)OnTF) protein possessed capability to bind both S. agalactiae and A. hydrophila in vitro. Taken together, the present study indicated that OnTF might be involved in host defense against bacterial infection in Nile tilapia.