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C-reactive protein (CRP) is an acute-phase protein that can be used as an early diagnostic marker for inflammation, which is also an evolutionarily conserved protein and has been identified from arthropods to mammals. However, the roles of CRP during the immune response of Nile tilapia (Oreochromis niloticus) remain unclear. In this study, a CRP gene from Nile tilapia (On-CRP) was identified, and its roles in response to bacterial infection were investigated in vivo or in vitro. On-CRP was found to contain an open reading frame of 675 bp, encoding a polypeptide of 224 amino acids with the conservative pentraxin domain. On-CRP shares more than 50% of its identity with other fish species, and 30% of its identity with mammals. The transcriptional level of On-CRP was most abundant in the liver and its transcripts can be remarkably induced following Streptococcus agalactiae and Aeromonas hydrophila infection. Furthermore, in vitro analysis indicated that the recombinant protein of On-CRP improved phagocytic activity of monocytes/macrophages, and possessed a bacterial agglutination activity in a calcium-dependent manner. Both in vivo and in vitro experiments indicated that On-CRP could promote inflammation and activate the complement pathway. However, a direct relationship between CRP and several immune pathways could not be confirmed. The present data lays a theoretical foundation to further explore the mechanism of how CRP protects fish against bacterial infection.