Abstract: Defensin is one of the most important antimicrobial peptides (AMPs) that participate in invertebrate innate immunity against invading pathogens. In the present study, a novel defensin was identified in small abalone (Haliotis diversicolor) that was denoted as HdDef1 by using RNA-seq and RACE techniques. The HdDef1 cDNA contained a 201 bp open reading frame (ORF) encoding 66 amino acids including a signal peptide of 18 amino acids and a mature peptide of 48 amino acids. The mature peptide of HdDef1 shared common features of AMPs, such as lower molecular mass, net positive charge (+1) and high hydrophobic residue ratio (45%). In addition, six cysteines in the mature peptide were arranged in the pattern of C-X₁₆-C-X₃-C-X₉-C-X₄-C-X₁-C and stabilized the α-helix/β-sheet motif (CSαβ) with three disulfide bonds (C1-C4, C2-C5 and C3-C6) in the predicted tertiary structure. Moreover, by comparision with the similar three-dimensional structure of Anopheles gambiae defensin and phylogenetic analysis, it is suggested that HdDef1 might be a new member of the arthropod defensin family. Quantitative real-time PCR analysis reveals that HdDef1 transcripts were expressed constitutively in intestine, head, gill, hepatopancreas, mantle and foot, with the highest level in hepatopancreas. After being challenged with Vibrio harveyi, HdDef1 transcripts were induced significantly in hepatopancreas. The results indicate that HdDef1 might have an important function in host defense against invasive pathogenic bacteria, but its antimicrobial activity at protein level needs further study.