Background and Purpose N-docosahexaenoylethanolamine (DHEA) is the ethanolamine conjugate of the long-chain polyunsaturated n-3 fatty acid docosahexaenoic (DHA; 22: 6n-3). Its concentration in animal tissues and human plasma increases when diets rich in fish or krill oil are consumed. DHEA displays anti-inflammatory properties in vitro and was found to be released during an inflammatory response in mice. Here, we further examine possible targets involved in the immune-modulating effects of DHEA. Experimental Approach Antagonists for cannabinoid (CB)1 and CB2 receptors and PPAR¿ were used to explore effects of DHEA on NO release by LPS-stimulated RAW264.7 cells. The possible involvement of CB2 receptors was studied by comparing effects in LPS-stimulated peritoneal macrophages obtained from CB2-/- and CB2+/+ mice. Effects on NF-¿B activation were determined using a reporter cell line. To study DHEA effects on COX-2 and lipoxygenase activity, 21 different eicosanoids produced by LPS-stimulated RAW264.7 cells were quantified by LC-MS/MS. Finally, effects on mRNA expression profiles were analysed using gene arrays followed by Ingenuity® Pathways Analysis. Key Results CB1 and CB2 receptors or PPARs were not involved in the effects of DHEA on NO release. NF-¿B and IFN-ß, key elements of the myeloid differentiation primary response protein D88 (MyD88)-dependent and MyD88-independent pathways were not decreased. By contrast, DHEA significantly reduced levels of several COX-2-derived eicosanoids. Gene expression analysis provided support for an effect on COX–2-mediated pathways. Conclusions and Implications Our findings suggest that the anti-inflammatory effects of DHEA in macrophages predominantly take place via inhibition of eicosanoids produced through COX-2.