Macrophages play a key role in obesity-induced inflammation. Omega-3 polyunsaturated fatty acids (v-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert anti-inflammatory functions in both humans and animal models, but the exact cellular signals mediating the beneficial effects are not completely understood. We previously found that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. Here we aim to determine whether v-3 PUFAs antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway. Treatment of v-3 PUFAs suppresses lipopolysaccharide (LPS)-induced cytokine expression in macrophages. Luciferase reporter assays, electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) assays show that treatment of macrophages with v-3 PUFAs significantly inhibits LPS-induced NF-kB signaling. Interestingly, DHA also increases expression, phosphorylation and activity of the major isoform a1AMPK, which further leads to SIRT1 overexpression. More importantly, DHA mimics the effect of SIRT1 on deacetylation of the NF-kB subunit p65, and the ability of DHA to deacetylate p65 and inhibit its signaling and downstream cytokine expression require SIRT1. In conclusion, v-3 PUFAs negatively regulate macrophage inflammation by deacetylating NF-kB, which acts through activation of AMPK/SIRT1 pathway. Our study defines AMPK/SIRT1 as a novel cellular mediator for the anti-inflammatory effects of v-3 PUFAs.