Hepcidin is a cysteine-rich antimicrobial peptide that plays an important role in fish immunity. In the current study, we report a novel isoform of hepcidin (Jd-Hep) from Sin croaker, Johnius dussumieri, with an open reading frame (ORF) of 258 nucleotide bases that encodes 85 amino acids containing a signal peptide (24 amino acids), a prodomain (35 amino acids) and a biologically active mature peptide (26 amino acids). Phylogenetic tree analysis showed that J. dussumieri hepcidin belonged to the HAMP2 cluster of hepcidin. The tissue distribution showed that the expression of hepcidin was highest in the liver in wild-caught J. dussumieri. The mature peptide mJd-Hep was recombinantly expressed in a prokaryotic host, E. coli Rosetta-gami™B (DE3) pLysS cells, and the peptide was isolated and purified. The recombinant peptide, rJd-Hep, exhibited notable antibacterial activity against aquatic pathogens such as Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio proteolyticus, and Vibrio fluvialis. The mode of action of the peptide was proven to be membrane-based (pore formation and depolarization). The rJd-Hep was found to be non-hemolytic to hRBCs and non-cytotoxic to the mammalian cell line. The peptide showed 85% growth inhibition of cancer cell line, MCF-7. These findings expand our knowledge of the potential application of hepcidin in aquaculture as a therapeutic agent.