The extracellular zinc metalloprotease (EmpA) is a putative pathogenic factor involved in the invasive process of the fish pathogen Vibrio anguillarum. It is synthesized as a 611-amino-acid preproprotease. The gene encoding EmpA (empA) has already been cloned and sequenced. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli BL21(DE3). Recombinant EmpA with His-tag was purified in a single step with a His-binding Ni-affinity column to a purity > 95%. In addition, proteolytic activity, cytotoxicity, fish pathogenicity, and solubility of the recombinant protein were determined.