The shrimp multifunctional protein alpha-2-macroglobulin (A2M) is abundantly expressed in plasma, highly up-regulated upon microbial infection and involved in several immune pathways such as blood clotting system, phagocytosis and melanization. Herein, the function of LvA2M from Litopenaeus vannamei on the prophenoloxidase (proPO) system is reported. The recombinant (r)LvA2M produced strongly and specifically inhibited trypsin and the PO activity in shrimp plasma in a dose-dependent manner. Silencing of LvA2M led to an increase in the PO activity in shrimp plasma although the expression of proPO-associated genes, proPO-activating enzyme (PPAE) and prophenoloxidase (proPO) but not the proPO-activating factor (PPAF) was down-regulated. In Vibrio parahaemolyticus AHPND-infected shrimp, the LvA2M activity was suppressed in an early phase of infection while the PO activity was increased. Thus, the proPO-activating system was regulated by the LvA2M.