Tumor necrosis factor (TNF)-alpha is elevated during the acute phase of Kawasaki disease (KD), which damages vascular endothelial cells to cause systemic vasculitis. In the current study, we investigated the potential role of cordycepin on TNF alpha expression in both lipopolysaccharide (LPS)-stimulated macrophages and ex vivo cultured peripheral blood mononuclear cells (PBMCs) of KD patients. We found that cordycepin significantly suppressed LPS-induced TNF alpha expression and production in mouse macrophages (RAW 264.7 cells and bone marrow-derived macrophages (BMDMs)). Meanwhile, cordycepin alleviated TNF alpha production in KD patients' PBMCs. PBMCs from healthy controls had a much lower level of basal TNF-alpha content than that of KD patients. LPS-induced TNF-alpha production in healthy controls' PBMCs was also inhibited by cordycepin. For the mechanism study, we discovered that cordycepin activated AMP-activated protein kinase (AMPK) signaling in both KD patients' PBMCs and LPS-stimulated macrophages, which mediated cordycepin-induced inhibition against TNF alpha production. AMPK inhibition by its inhibitor (compound C) or by siRNA depletion alleviated cordycepin's effect on TNF alpha production. Further, we found that cordycepin inhibited reactive oxygen species (ROS) production and nuclear factor kappa B (NF-kappa B) activation in LPS-stimulate RAW 264.7 cells or healthy controls' PBMCs. PBMCs of KD patients showed higher basal level of ROS and NF-kappa B activation, which was also inhibited by cordycepin co-treatment. In conclusion, our data showed that cordycepin inhibited TNF alpha production, which was associated with AMPK activation as well as ROS and NF-kappa B inhibition. The results of this study should have significant translational relevance in managing this devastating disease.