Eicosadienoic acid (Δ11,14-20:2; EDA) is a rare, naturally occurring n-6 polyunsaturated fatty acid (PUFA) found mainly in animal tissues. EDA is elongated from linoleic acid (LA), and can also be metabolized to dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA), and sciadonic acid (Δ5,11,14-20:3; SCA). Although, the metabolism of EDA has been extensively studied, there are few reports regarding how EDA might affect inflammatory processes. The objective of this study was to determine the effect of EDA on the n-6 PUFA composition and inflammatory response of murine RAW264.7 macrophages to lipopolysaccharide (LPS). EDA was taken up rapidly by macrophages and metabolized to SCA, and the percentages of both fatty acids increased in cellular phospholipids in a dose-dependent manner. The incorporation of EDA into macrophage lipids increased the proportions of LA, DGLA, and AA as well, and reduced the proportion of total monounsaturated fatty acids. When LPS were applied to the macrophages, EDA decreased the production of nitric oxide (NO), and increased that of prostaglandin E(2) (PGE(2)) and tumor necrotic factor-α. The modulation of NO and PGE(2) was due, in part, to the modified expression of inducible nitric oxide synthase and type II cyclooxygenase. The differential effects of EDA on pro-inflammatory mediators might attribute to the negative feedback mechanism associated with prolonged inflammation. Furthermore, EDA was a weaker pro-inflammatory agent than LA, and not as anti-inflammatory as SCA. This study shows that EDA can modulate the metabolism of PUFA and alter the responsiveness of macrophages to inflammatory stimulation.