Macrophage migration inhibitory factor (MIF) is evolutionarily ancient and has been found across kingdoms including animals, plants and bacteria. In mammals, MIF has been suggested as chemokine-like cytokine with enzymatic activities, which plays a critical role in inflammatory response against pathogen infections. In this study, we cloned a MIF-like (named after AjMIF) gene for the first time in Japanese eel, Anguilla japonica. The AjMIF precursor processes MIF signature sequence motif, Cys₅₇-Ala-Leu-Cys₆₀, for its thiol-protein oxidoreductase activity, and several conserved residues which are critical for its isomerase activity, such as Pro₂ and Cys₈₁. Expression analysis showed that AjMIF is expressed in various organs / tissues with the highest in liver, followed by middle kidney and intestine. The expression level of AjMIF was significantly increased in head kidney, middle kidney and swim bladder after challenged with lipopolysaccharides at 8 hours post-injection (hpi). Significant increase in AjMIF expression was also observed in gill, skin and intestine at 8 hpi following stimulation with polyinosinic-polycytidylic acid. In addition, significant increase of AjMIF mRNA in gill and intestine was observed at 8 hpi, and in gill at 16 and 24 hpi, and in skin at 24 hpi when infected with Edwardsiella tarda. Furthermore, pH-dependent tautomerase activity of AjMIF has also been found by a recombinant rAjMIF using a prokaryotic expression system. Our results showed that 1 nmol of rAjMIF exhibited 2.6 U of tautomerase activity at pH 6.2, but 36.6 U at pH 8.0. Overall, our study provides a basis for future research aiming at a better understanding the functions of MIF in fish immune system.