Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, in addition to melanin, the proPO system produces reactive intermediates that are not only harmful to the invading microbes but also to the host cells. Thus, the proPO system must be tightly regulated by several inhibitors. Previously, a melanization inhibition protein from the black tiger shrimp Penaeus monodon, PmMIP, has been identified and preliminarily characterized. In this study, we investigate the function of PmMIP in the regulation of the proPO system in shrimp. When challenged with the bacterium Vibrio harveyi, the expression of PmMIP transcripts in gills was down-regulated dramatically at 24 h but recovered after 48 h post infection (hpi), while the PmMIP protein level in shrimp plasma was decreased at 6 hpi but recovered at 24 hpi. Double-stranded RNA-mediated gene silencing of PmMIP suppressed both PmMIP transcriptional and translational levels and resulted in increased hemolymph phenoloxidase and proteinase activities compared to controls injected with GFP dsRNA or NaCl. Furthermore, the recombinant PmMIP protein successfully expressed in Escherichia coli was able to inhibit hemolymph PO activity by 50%. These results suggested that PmMIP was involved in the proPO system by acting as a negative regulator and interfering with hemolymph proteinase activity.