The skin mucus of the great sculpin Myoxocephalus polyacanthocephalus showed both antibacterial and L-amino acid oxidase (LAO) activities. Antibacterial LAOs were purified from the skin mucus of the M. polyacanthocephalus by column chromatography and named MPLAO1, MPLAO2, and MPLAO3, based on the order of elution by ion-exchange high performance liquid chromatography. cDNA cloning of MPLAO3 revealed that the full-length cDNAwas 2659 bp and encoded the signal peptide (Met1-Ala26) and the mature protein (Val28-Phe520). A homology search using the BLAST program revealed that MPLAO3 shared sequence identity with LAO family proteins, and had 74% identity with the antibacterial LAO from the skin mucus of the rockfish Sebastes schlegeli. MPLAO3 catalyzed the oxidation of only L-lysine with a Km of 0.16 mM. MPLAO3 exhibited potent antibacterial activity against both Gram-positive and Gramnegative bacteria, and was most active against Aeromonas salmonicida JCM7874 with a minimum inhibitory concentration of 0.02 microg/mL. The antibacterial activity was attributable to H2O2, because the activity was completely lost in the presence of catalase. The antibacterial LAOs may be involved in the innate immunity of the great sculpin M. polyacanthocephalus skin.