Cathelicidins, one family of antimicrobial peptides, play important roles against infections in animals. In this study, a cDNA sequence coding for cathelicidin was cloned from constructed liver cDNA library of ayu, Plecoglossus altivelis. The deduced ayu cathelicidin (aCATH) has a 20 amino acid residue signal peptide, a conserved cathelin domain of 110 amino acid residues and a mature antimicrobial peptide of 61 amino acid residues. Sequence comparison and phylogenetic tree analysis confirmed aCATH as a distinct member of fish cathelicidins. Real-time quantitative PCR revealed that the aCATH transcripts dramatically increased in various tissues after bacterial infection. Subsequently, aCATH was prokaryotic expressed and purified. Western blot and mass spectrometry revealed that aCATH was cleaved at residue Ile130-Arg131 by human neutrophil elastase to release the mature antimicrobial peptide. The mature peptide of aCATH was chemically synthesized and exhibited potent antimicrobial activity. Thus, aCATH may play an important role in the innate immunity of ayu, and this work enriches our knowledge in fish antimicrobial peptides.