AbstractNiemann–Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1‐deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E2 compared with wild‐type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A2α (cPLA2α) and cultivation in lipoprotein‐deficient medium. There was no difference in the expression of both cyclooxygenase‐1 and ‐2 between NPC cells and wild‐type cells. U18666A, a cholesterol transport‐inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A‐induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA2α knockdown L929 cells. These results suggest that cPLA2α‐AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells. J. Cell. Physiol. 227: 2847–2855, 2012. © 2011 Wiley Periodicals, Inc.