Polyunsaturated fatty acids (PUFA) can modulate the immune response, however the mechanism by which they exert this effect remains unclear. Previous studies have clearly demonstrated that the cis-9, trans-11 isomer of conjugated linoleic acid (c9,t11-CLA), found predominantly in beef and dairy products, can modulate the response of immune cells to the toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). This study aimed to investig ate further the mechanism by which these effects are mediated. Treatment of macrophages with c9,t11-CLA significantly decreased CD14 expression and partially blocked its association with lipid rafts following stimulation with LPS. Furthermore the c9,t11-CLA isomer inhibited both nuclear facto r- κ B (NF- κ B) and IRF3 activation following TLR4 ligation while eicosapentaenoic acid (EPA) only suppressed NF- κ B activation. Given that the ability of LPS to activate IRF3 downstream of TLR4 depends on internalisation of the TLR4 complex and involves CD14, we examined TLR4 endocytosis. Indeed the internalisation o f TLR4 to early endosomes following activation with LPS was markedly inhibited in c9,t11-CLA treated cells. These effects were not seen with the n-3 fat ty acid, EPA, which was used as a comparison. Our data demonstrates that c9,t11-CLA inhibits IRF3 activation via its effects on CD14 expression and localisation. This results in a decrease in the endocytosis of TLR4 which is necessary for IRF3 activation, revealing a novel mechanism by which this P UFA exerts its anti-inflammatory effects. ; PUBLISHED ; This work was supported by funds from the Irish Research Council for Science, Engineering and Technology and Science Foundation Ireland