Despite having a similar structure, various conjugated linoleic acid (CLA) isomers have a distinct gene expression pattern in RAW 264.7 (RAW) cells, a mouse macrophage cell line. Among the 5 CLA isomers tested [9cis(Z),11trans(E)-, 9Z,11Z-, 9E,11E-, 10E,12Z-, and 11Z,13E-CLA], only 9E,11E-CLA induced the endogenous antiinflammatory molecule, interleukin (IL)-1 receptor antagonist (IL-1Ra), in RAW cells. In this study, the mechanism and effects of IL-1Ra regulation by 9E,11E-CLA in RAW cells was studied in detail. 9E,11E-CLA induced IL-1Ra in a dose- and time-dependent manner, whereas it decreased lipopolysaccharide (LPS)-induced IL-1alpha, IL-1beta, and IL-6 mRNA levels and protein levels. To determine the importance of IL-1Ra in the antiinflammatory effects of this particular CLA isomer, IL-1Ra protein levels were repressed in RAW cells using small interference RNA inhibitor expression. In the presence of IL-1Ra small interference RNA, the induction of this molecule was ablated, as was the decrease of LPS-induced IL-1alpha and IL-6 mRNA levels by 9E,11E-CLA. The IL-1Ra increase due to this CLA isomer was transcriptionally regulated, although there was no response element(s) affected by 9E,11E-CLA in the first 1.5 kb of the IL-1Ra promoter. The phosphoinositide 3-kinase inhibitors, LY294002 and the mammalian target of rapamycin inhibitor rapamycin, abolished the IL-1Ra induction by 9E,11E-CLA, whereas other kinase inhibitors did not affect this response. Taken together, 9E,11E-CLA exerts unique antiinflammatory effects by increasing an endogenous repressor of IL-1 signaling.