Antimicrobial peptides (AMPs) are important mediators of the immune response against bacteria and cysteine-rich peptide hepcidin is a 20-26 residues member with functions in iron regulation and antimicrobial activity. Here, we have identified two different types of hepcidin cDNA from the Black rockfish, Sebastes schlegelii, by EST analysis. Both hepcidin genes (hepcidin I and II) consist of two introns and three exons that encode a prepropeptide (88 amino acids). A TATA box and several consensus-binding motifs for transcription factors were found in the upstream of the transcriptional start site. Semi-quantitative RT-PCR analysis suggested that hepcidin I transcripts were detected in various tissues, while hepcidin II was only expressed in the liver. During the bacterial challenge with the fish pathogen, Streptococcus iniae, two hepcidin genes were differentially expressed. Hepcidin I and II dramatically increased at 24 h post-injection, then gradually declined at 3 days in hepcidin II, while hepcidin I expression continued at 3 days after challenge.