Phenoloxidase (PO) plays a critical role in insect immune reactions especially to form melanotic encapsulation and phagocytosis by hemocytes. PO is an active form of prophenoloxidase (PPO) after proteolytic cleavage by serine proteinase(s). It has been suggested that eicosanoids are implicated in PPO activation in the beet armyworm, Spodoptera exigua. However, it is not clear how eicosanoids mediate the reaction cascade of PPO activation. This study analyzed the PPO activation mediated by eicosanoids at both transcriptional and post-transcriptional levels. A cDNA encoding PPO (SePPO) was cloned from the hemocytes of S. exigua and its putative amino acid sequence shared homology with PPO-2 of other lepidopteran insects. Its expression was specific only to hemocytes and inducible in response to bacterial challenge. Eicosanoid biosynthesis inhibitors did not influence the gene expression of SePPO. Most SePPO proteins were located in a specific hemocyte type, oenocytoids, which were subjected to cell rupture to release the cellular contents in response to bacterial challenge. There was a significant negative correlation between PO activity and intact oenocytoid density. Interestingly, this cell rupture to release SePPO from oenocytoids was significantly inhibited in the larvae infected with the phospholipase A2-inhibiting bacterium, Xenorhabdus nematophila, which was resumed on addition of eicosanoid biosynthesis precursor, arachidonic acid. Furthermore, oenocytoids exposed to eicosanoid biosynthesis inhibitors such as dexamethasone and bromophenacyl bromide showed significant reduction in cell rupture. Prostaglandins, not lipoxygenase products appeared to be implicated in the cell rupture. These results indicate that eicosanoids mediate SePPO activation only at the post-transcriptional level by inducing release of PPO from oenocytoids through cell rupture.