Eicosapentaenoic acid (EPA) is an omega-3 (omega-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-alpha-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-alpha induced MMP-9 expression by NF-kappaB-dependent pathway. Pretreatment of EPA inhibited TNF-alpha-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IkappaB-alpha phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-kappaB. EPA inhibited TNF-alpha-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKalpha-dependent event. Taken together, we demonstrate that EPA inhibits TNF-alpha-induced MMP-9 expression through inhibition of p38 and Akt activation.