We previously reported that ONO-AE-248, a selective EP3 receptor agonist, has been shown to cause neutrophil death without the typical features of apoptosis and necrosis. However, the mechanism of the neutrophil death is unclear. By using West blotting, flow cytometry (FACS) and confocal laser scanning microscopy (CLSM), we investigated the cellular signal transduction pathways of the neutrophil death. The research results showed that the neutrophil death induced by ONO-AE-248 did not show the morphologic changes of apoptosis and was not associated with the activity of caspase-3, caspase-8, and phosphorylation of p38-MAPK. However, impairment of mitochondria transmembrane potential has been found during the process of cell death. These findings suggested that ONO-AE-248 induced a non-apoptotic programmed cell death of neutrophils through partially mitochondria signaling transduction pathway.