Olive oil contains high amounts of oleic acid (OA). Although OA has been described to inhibit inflammatory processes, the effects of olive oil on cellular mechanisms remain poorly understood. Therefore, we compared the effects of major fatty acids (FA) from olive oil with those of olive oil extracts (OOE) on inflammatory mediators and alterations in the cellular phospholipid composition in murine macrophages. Upon treatment with different OOE, FA compositions of lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages were analyzed using gas chromatography. Olive oil extracts and OA significantly reduced the LPS-induced expression of inducible nitric oxide synthase (iNos), cyclooxygenase (Cox2), and interleukin-6 mRNA. In addition, a significant decrease in Cox2 and iNos protein expression was observed. The formation of nitric oxide was significantly reduced, while the formation of prostaglandin (PG) E₂ from arachidonic acid significantly increased after treatment with OOE or OA. The latter was associated with a shift in the phospholipid FA composition from arachidonic acid to OA, resulting in an elevated availability of arachidonic acid. Together, OOE and OA mediate anti-inflammatory effects in vitro but increase the release of arachidonic acid and hereinafter PGE₂, likely due to elongation of OA and competitive incorporation of fatty acids into membrane phospholipids.