Cytosolic lipid droplets (LDs), which are now recognized as multifunctional organelles, accumulate in leukocytes under various inflammatory conditions. However, little is known about the characteristic features of LDs in neutrophils. In this study, we show that perilipin-3 (PLIN3; formerly called TIP47) is involved in LD formation and the inflammatory response in HL-60-derived neutrophils. HL-60, a promyelocytic cell line, was differentiated into neutrophils via treatment with all-trans retinoic acid. After differentiation, cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g-LPS), a major pathogen in adult periodontitis. When HL-60-derived neutrophils were stimulated with P.g-LPS, LDs increased in both number and size. In the differentiated cells, PLIN3 was induced while PLIN1, PLIN2 and PLIN5 were not detected. PGE2 production and the PLIN3 protein level were increased by the P.g-LPS treatment of the cells in a dose-dependent manner. When PLIN3 was down-regulated with siRNA treatment, LDs essentially disappeared and the level of PGE2 secreted in the cell culture medium decreased by 65%. In addition, the suppression of PLIN3 repressed the PGE2 producing enzymes; i.e., microsomal PGE synthase-1, -2 and cyclooxygenase-2. These findings indicate that PLIN3 has a pivotal role in LD-biogenesis in HL-60-derived neutrophils, and that PLIN3 is associated with the synthesis and secretion of PGE2.