Long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) have been shown to modulate the immune response and have therapeutic effects in inflammatory disorders. PUFA are also peroxisome proliferators-activator receptor-gamma (PPARγ) ligands; a family of ligand-activated transcription factors, which when activated antagonise the pro-inflammatory capability of nuclear factor κB (NF-κB). PPARγ plays a role in dendritic cell (DC) maturation and n-3 PUFA have been shown to affect DC maturation by decreasing activation of NF-κB. While n-3 PUFA can function as PPAR ligands, it is not known whether the NF-κB-mediated immunomodulatory properties of n-3 PUFA are PPARγ-dependent. In this study we examined whether the immunomodulatory effects of n-3 PUFA on DC activation were mediated through activation of PPARγ. Treatment of murine bone marrow derived DCs with docosahexaenoic acid (DHA; 25 μM) and eicosapentaenoic acid (EPA; 25 μM) attenuated LPS-induced DC maturation. This was characterised by suppression of IL-12 production and expression of CD40, CD80, CD86 and MHC II and enhanced production of IL-10 and expression of IL-10R. This was coincident with enhanced PPARγ expression, suppressed NF-κB activity and increased the physical interaction and cellular colocalization between NF-κB with PPARγ. To understand the functional implication of the physical association of PPARγ with NF-κB, we determined whether the specific PPARγ inhibitor, GW9662 could abolish the anti-inflammatory effect of n-3 PUFA Inhibiting PPARγ did not impede the NF-κB-mediated anti-inflammatory cytokine profile induced by EPA and DHA alone. Thus n-3 PUFA activate PPARγ and interact with NF-κB in DC. However, the anti-inflammatory effects of EPA and DHA on DCs are independent of PPARγ.