Phenoloxidase is the key enzyme in insect innate immune system. It plays an important role in cellular immunity during melanotic encapsulation and phagocytosis by hemocytes. Phenoloxidase is an active form after proteolytic cleavage of prophenoloxidase by serine protease. It has been suggested that eicosanoids are implicated in the pPO activation in the beet armyworm, Spodoptera exigua. However, it is not clear how eicosanoids are involved in the reaction cascade of pPO activation. This study was designed to analyze the pPO activation in both transcription and post-transcriptional controls. A cDNA of pPO was cloned from the hemocytes of S. exigua and called as SePPO. The amino acid sequence of SePPO shared homology with PPO-2 with other insects. A RT-PCR analysis indicated that SePPO expressed only in hemocytes and inducible in response to a bacterial challenge. Its gene expression did not affect with eicosanoid biosynthesis inhibitors. Most pPO appeared to be synthesized in oenocytoid hemocytes, which were released into the hemocoel by cell rupture in response to the bacterial challenge. However, this SePPO release was prevented by the injection of phospholipase A2-inhibiting bacterium, Xenorhabdus nematophila, which was rescued by addition of eicosanoid precursor, arachidonic acid. Oenocytoids treated with eicosanoid biosynthesis inhibitors such as dexamethasone and bromophenacyl bromide reduced release of SePPO from the oenocytoids. These results suggest that eicosanoids are involved in release of SePPO from oenocytoid in post-transcriptional level.