Crustin antimicrobial peptides, identified in crustaceans, are hypothesized to have both antimicrobial and protease inhibitor activity based on their primary structure and in vitro assays. In this study, a reverse genetic approach was utilized to test the hypothesis that crustins are antimicrobial in vivo in response to bacterial and fungal challenge. Injection of double-stranded RNA specific to a 120-bp region of LvABP1, one of the most prominent crustin isoforms, yielded a significant reduction in the expression of both crustin mRNA and protein within the hemocytes. To test the role of crustins in the shrimp immune response, RNAi was first used to suppress crustin expression and animals were subsequently injected with low pathogenic doses of either Vibrio penaeicida or Fusarium oxysporum. A significant increase in mortality in crustin-depleted animals was observed in animals infected with V. penaeicida as compared to controls, whereas no significant change in shrimp mortality was observed following infection with F. oxysporum.