Docosahexaenoic acid (DHA) protects neural cells from stress-induced apoptosis. On the contrary, DHA exerts anticancer effects, and we have shown that DHA induces apoptosis in neuroblastoma, an embryonal tumor of the sympathetic nervous system. We now investigate the DHA metabolome in neuroblastoma using a targeted lipidomic approach in order to elucidate the mechanisms behind the DHA-induced cytotoxicity. LC-MS/MS analysis was used to identify DHA-derived lipid mediators in neuroblastoma cells. Presence of the 15-lipoxygenase enzyme was investigated using immunoblotting, and cytotoxic potency of DHA and DHA-derived compounds was compared using the MTT cell viability assay. Neuroblastoma cells metabolized DHA to 17-hydroxydocosahexaenoic acid (17-HDHA) via 17-hydroperoxydocosahexaenoic acid (17-HpDHA) through 15-lipoxygenase and autoxidation. In contrast to normal neural cells, neuroblastoma cells did not produce the anti-inflammatory and protective lipid mediators, resolvins and protectins. 17-HpDHA had significant cytotoxic potency, with an IC₅₀ of 3–6 μM at 72 h, compared to 12–15 μM for DHA. -Tocopherol protected cells from 17-HpDHA-induced cytotoxicity. DHA inhibited secretion of prostaglandin-E₂ and augmented the cytotoxic potency of the cyclooxygenase-2-inhibitor celecoxib. The cytotoxic effect of DHA in neuroblastoma is mediated through production of hydroperoxy fatty acids that accumulate to toxic intracellular levels with restricted production of its products, resolvins and protectins.—Gleissman, H., Yang, R., Martinod, K., Lindskog, M., Serhan, C. N., Johnsen, J. I., Kogner, P. Docosahexaenoic acid metabolome in neural tumors: identification of cytotoxic intermediates.