The piscidin (pis) family of potent antimicrobial peptides with broad-spectrum activity has an important role in innate host defence. We have identified and characterized two pis paralogues in Atlantic cod (pis1 and pis2), as well as a novel splice variant of pis2, termed pis2-β. Pis1 and pis2 genes have most likely originated from a recent duplication event, since they share the same four-exon structure with up to 91% identity at the intron level. The alternative transcript pis2-β is derived from intron retention and even if not translated it may regulate pis expression through nonsense mediated decay. In spite of their overall conservation, pis genes are being shaped by positive selection and pis1, pis2 and pis2-β code for structurally diverse mature peptides, which have different functional properties. Synthetic Pis1 displays antibacterial activity in the micromolar range against Gram-(+) and Gram-(-) bacteria, including the fish pathogens Vibrio anguillarum and Yersinia ruckeri. In contrast, synthetic Pis2 and Pis2-β have limited or no antibacterial activity, respectively, but exhibit more potent antiparasitic activity against Tetrahymena pyriformis. In adult cod, pis1 and pis2-β are constitutively expressed in immune-related organs, whereas pis2 is constitutively expressed in all tissues examined. Differential expression is also observed during embryonic development. In particular, pis2 and pis2-β are maternally inherited but pis1 transcripts are only present from gastrulation onwards. It was found that antigenic challenge with attenuated V. anguillarum induces a general down-regulation of all pis in head kidney, spleen and distal intestine, suggesting that they may be used as health indicators. Taken together, our data indicate that pis is an important component of the cod innate immune system. Moreover, the two pis paralogues have undergone structural diversification and it is likely that they play multifunctional roles in Atlantic cod.