The hepcidin gene is widely expressed in many fish species and functions as an antimicrobial peptide, suggesting that it plays an important role in the innate immune system of fish. In the present study, the Amatitlania nigrofasciata hepcidin gene (AN-hepc) was cloned from the liver and its expression during an immune response was characterized. The results of quantitative PCR and RT-PCR showed that the AN-hepc transcript was most abundant in the liver. The expression of AN-hepc mRNA was significantly increased in the liver, stomach, heart, intestine, gill and muscle but was not significantly altered in the spleen, kidney, brain or skin after lipopolysaccharide challenge. The synthetic AN-hepc peptide showed a wide spectrum of antimicrobial activity in vitro toward gram-positive and gram-negative bacteria. In particular, this peptide demonstrated potent antimicrobial activity against the aquatic pathogens Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, Aeromonas hydrophila and Streptococcus agalactiae. The in vivo bacterial challenge results demonstrated that the synthetic AN-hepc peptide significantly improved the survival rate of S. agalactiae- and V. vulnificus-infected zebrafish. Taken together, these data indicate an important role for AN-hepc in the innate immunity of A. nigrofasciata and suggest its potential application in aquaculture for increasing resistance to disease.