Long-term total parenteral nutrition (TPN) in children is often complicated by parental nutrition-associated liver disease and may even lead to liver failure. Recently, the addition of ω-3 fatty acids to TPN has been shown to reduce the risk of parental nutrition-associated liver disease. The purpose of this study was to explore the anti-inflammatory effects of ω-3 fatty acids (eicosapentaenoic acid [EPA]) to demonstrate the protection of the liver against hepatic steatosis and damage.Lipopolysaccharide (LPS) and prostaglandin E(2) (PGE(2)) were used to stimulate human macrophages and hepatocytes (THLE-3) to induce in vitro inflammatory condition. The cells were then incubated with either ω-3 (EPA) or ω-6 (arachidonic acid) fatty acids. Supernatants were collected at different time points for the measurement of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10) using enzyme-linked immunosorbent assay. Furthermore, pretreated macrophages by LPS stimulation and after incubation with EPA were added to prestimulated hepatocytes for the subsequent measurement of cytokine response.Eicosapentaenoic acid effectively reduced LPS-induced or PGE(2)-induced TNF-α and IL-6 expression, and increased IL-10 expression significantly when compared with arachidonic acid. Furthermore, supernatant collected after co-culturing EPA with macrophages also suppressed the levels of TNF-α and IL-6 in hepatocytes. This would suggest that EPA not only had an anti-inflammatory effect on macrophages and hepatocytes directly, it could indirectly reduce hepatocyte inflammation through activated macrophages.The addition of ω-3 fatty acids in TPN suppresses the inflammatory response via direct and indirect routes. The findings may help explain the clinical benefits of EPA in pediatric patients receiving long-term TPN.