The zinc metalloprotease EmpA is a virulence factor in the fish pathogen Vibrio anguillarum. Previous studies have shown that two strains of V. anguillarum regulate empA differently. Strain M93Sm exhibits protease activity only in the presence of fish gastrointestinal mucus, while protease activity is detected in NB10 culture supernatant under all stationary-phase conditions. In this study, we use real-time reverse transcription-PCR to show that even in conditions where no protease activity is detected, empA transcription occurs. Western blot analysis revealed that EmpA is secreted as a approximately 48-kDa proenzyme and that activation occurs extracellularly by the removal of a approximately 10-kDa peptide. The presence of stable extracellular pro-EmpA in M93Sm culture supernatants suggests that activation of EmpA is not autolytic.