Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix. In order to study the function of MMPs in the immune defense of Apostichopus japonicus, the full-length cDNA sequence of the matrix metalloproteinase 16 gene, named Aj-MMP-16, was cloned using RACE method. The sequence characteristics and function of Aj-MMP-16 were preliminarily analyzed. The results showed that the full-length cDNA of this gene was 2 976 bp, including a 5' non-coding region of 342 bp, a 3' non-coding region of 963 bp, and an open reading frame (ORF) of 1 671 bp encoding 557 amino acids. The predicted molecular weight of Aj-MMP-16 protein was 63.11 ku and isoelectric point was 4.79. Functional domain analysis revealed the typical MMPs family protein structure of Aj-MMP-16 including N-terminal propeptide region, hinge region, catalytic region, hemopexin-like domain and transmembrane region. Multiple sequence alignment and phylogenetic analysis showed that Aj-MMP-16 shared a certain degree of conservatism with MMPs of other species and had the highest identity with MMP-16 of Strongylocentrotus purpuratus. Quantitative real time PCR showed that the Aj-MMP-16 mRNA was expressed in all tissues of A. japonicus, and the expression levels were from high to low in the order of respiratory tree, intestine, coelomocytes, tube feet, muscle and body wall. At different stages of skin ulceration syndrome progression, the expression of Aj-MMP-16 mRNA in ulcerate body wall was significantly higher than that of the normal body wall. After the pathogenic bacteria challenge, Aj-MMP-16 mRNA expression increased significantly in coelomocytes. The results suggested that Aj-MMP-16 may play important roles in visceral regeneration, inflammation and immune response in sea cucumber.