Abstract: IFN-γ is a cytokine that is critical for innate and adaptive immunity against viral, some bacterial and protozoal infections. In this study, an interferon-γ gene, named AjIFN-γ, was cloned and characterized from Japanese eel, Anguilla japonica. The AjIFN-γ shared some common features with its vertebrate homologues including a 4-exon/3-intron gene structure, a typical IFN-γ characteristic motif and a predicted nuclear localization site in the predicted protein. AjIFN-γ mRNA could be detected in all the tested tissues from healthy Japanese eel with the highest in liver, followed by skin and head kidney by real-time RT-PCR. A significantly increased expression of AjIFN-γ could be found in gill, head kidney, trunk kidney and (or) spleen post intraperitoneal injection with Poly I:C or Edwardsiella tarda, which indicated a role in defense of Japanese eel against both viruses and bacteria. Furthermore, luciferase reporter assay demonstrated that the sequence from –240 bp to +136 bp in the 5′ flanking region of AjIFN-γ gene was essential for initiating the transcription of AjIFN-γ, and the sequence form –1062 bp to –814 bp may contain some positive transcriptional regulatory elements while the sequence from –1252 bp to –1062 bp may contain negative transcriptional regula-tory elements. This study provided the basis for further investigation of the expanding functions of IFN-γ molecules in immunity and other physiological processes in teleost and other animals.