In order to study the role of hemojuvelin (hjv) in the process of resisting pathogen infection and maintaining its iron homeostasis in teleost fish, the present study amplified the open reading frame (ORF) of Oreochromis niloticus hjv (Onhjv), and analyzed its distribution pattern in various tissues of healthy tilapia and its related role in resisting pathogen infection and regulating iron homeostasis. The ORF of Onhjv was 1 248 bp, encoding 415 amino acids, which was conserved among different species. The results of quantitative real-time PCR (qRT-PCR) showed that Onhjv was widely distributed in all detected tissues of O. niloticus, and the highest expression level was found in liver. The expression of Onhjv was significantly up-regulated in the liver, spleen, intestine and gills after being challenged by Streptococcus agalactiae or Aeromonas hydrophila. The Onhjv expression in monocytes/macrophages (MO/Mø) and hepatocytes was also up-regulated after being stimulated by these two pathogens. In addition, the expression of Onhjv also showed an up-regulation trend in the liver, spleen, intestine, and gills, and head kidney MO/Mø and hepatocytes after stimulation with 1 μmol/L or 10 μmol/L FeCl₃ solution. After stimulation with recombinant O. niloticus IL-6 protein [(r)OnIL-6)], the expression of Onhjv in head kidney MO/Mø was significantly up-regulated, indicating that inflammatory factors could promote the expression of Onhjv. In this study, the results indicate that the hemojuvelin in O. niloticus plays an important role in host resistance to pathogen infection and maintenance of iron homeostasis.