Penaeidins are members of a special family of antimicrobial peptides existing in penaeid shrimp and play an important role in the immunological defence of shrimp. Here, we report one penaeidin with a putative isotype newly cloned from fleshy prawn Fenneropenaeus chinensis. The penaeidin open reading frame encodes a 79 amino acid peptide while two exons and an intron were identified within the 1126 bp genomic sequence of Fenchipenaeidin 5. Phylogenetic analysis and sequence comparison with other known penaeidins suggest the new gene belongs to a novel subfamily of penaeidins and the two isoforms were named Fenchi-penaeidin 5-1 and 5-2, respectively. Fenchi-penaeidin 5 mRNA was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in heamocytes, heart, gill, intestine and ovary. Bacterial challenge resulted in mRNA up-regulation, inducing expression in hepatopancreas and stomach. Fenchi-penaeidin 5-1 was also expressed in Pichia pastoris, and recombinant Fenchi-penaeidin 5-1 exhibited activities against Gram-positive and -negative bacteria and fungi.