Large yellow croaker (Pseudosciaena crocea) is one of the most important marine cultured fish in China. Acidic extracts of five tissues of large yellow croaker showed strong anti-Vibrio alginolyticus activity. Acidic extract of head kidney tissue was subjected to heat-treatment in boiling water, and solid-phase extraction on Sep-Pak C18 cartridge. It was found that the antibacterial substances were heat stable, and 20% acetonitrile effluent exhibited strong antibacterial activity. Active extract was further applied to Sephadex G-25 gel permeation chromatography and StableBond C18 RP-HPLC. An antibacterial peptide with a single peak was obtained. The results of amino acid sequencing and MALDI-TOF MS suggested that the peptide was RCRFCCRCCPRMRGCGICCRF with an observed molecular mass of 2523.2 Da. BLAST searching suggested that the purified antibacterial peptide was the mature peptide section of the hepcidin preproprotein presumed from cDNA of large yellow croaker, thus designated hepcidin-Pl. Hepcidin-P1 exhibited strong antibacterial activity against four marine vibrios.