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Serine proteases and their inhibitors play vital roles in biological processes. Serine protease inhibitors, including Kunitz-type protease inhibitors play important roles not only in physiological process (i.e. blood clotting and fibrinolysis) but also in immune responses. In this study, we characterized a Kunitz-type protease inhibitor, designated MjKuPI, from kuruma shrimp Marsupenaeus japonicus. An expression profile showed that MjKuPI was mainly expressed in hemocytes. Immunostaining revealed that some hemocytes expressed MjKuPI (MjKuPI+ hemocytes) and others did not (MjKuPI− hemocytes). Injection of shrimp with Vibrio penaeicida and white spot syndrome virus (WSSV) upregulated the mRNA level of MjKuPI, and a flow cytometry analysis revealed that the proportion of MjKuPI+ hemocytes increased significantly 24 h after injection. Together, these results suggest that MjKuPI and MjKuPI+ hemocytes have a role in the innate immune system of kuruma shrimp.MjKuPI mRNA level increased significantly in WSSV and Vibrio penaeicida challenged shrimps.Immunostaining results showed that only one type of hemocytes expressed MjKuPI protein.The number of MjKuPI expressed hemocytes increased significantly in challenged shrimps.