The phenoloxidase (PO) activation system plays an important role in insect innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating protease (PAP), which is the direct activator of prophenoloxidase (proPO). Despite their importance in the insect PO activation system, content of studies is limited. In this article, we identify two complementary DNAs (cDNAs), PxPAPa and PxPAPb, encoding possible PAPs, from immunized larval hemocytes of the diamondback moth, Plutella xylostella (L.), by RACE method. PxPAPa is 1,149-bp long and encodes a 382-residue open reading frame (ORF) with a predicted 17-residue signal peptide, a clip domain, and a Tryp_Spc domain. PxPAPb is 1,650-bp long and encodes a 440-residue ORF with a predicted 20-residue signal peptide, two clip domains, and a Tryp_Spc domain. PxPAPa and PxPAPb have a high sequence similarity to Manduca sexta (L.) PAP1 and PAP3, respectively. We also examined the transcript patterns of PxPAPa, PxPAPb, and pxPAP3, another clip-domain serine protease gene, response to different microbial challenges by using real-time quantitative polymerase chain reaction. The results show that the transcript abundance of PxPAPa is significantly increased by Micrococcus luteus and Escherichia coli but not Candida albicans. PxPAPb is induced only by Mi. luteus, whereas pxPAP3 could be induced by all the microbes in the test, but the transcript patterns of Mi. luteus, E. coli, and C. albicans are completely different. This study provides new insights into the molecular events that occur during the immune response, particularly melanization cascade that is involved in encapsulation and nodulation of pathogen or parasite invaders via hemocytes in host insects.