The fuel sensing enzyme AMP-activated protein kinase (AMPK) enhances processes that generate ATP when stresses such as exercise or glucose deprivation make cells energy deficient. We report here a novel role of AMPK, to prevent the activation of NF-κB in endothelial cells exposed to the fatty acid palmitate or the cytokine TNF-α. Incubation of cultured human umbilical vein endothelial cells (HUVEC) with elevated levels of palmitate (0.4mM) increased NF-κB reporter gene expression by 2- to 4-fold within 8h and caused a 7-fold increase in VCAM-1 mRNA expression at 24h. In contrast, no increase in reporter gene expression was detected for AP-1, glucocorticoid-, cyclic AMP-, or serum response elements. Similar increases in NF-κB activation and VCAM-1 expression were not observed in cells incubated with an elevated concentration of glucose (25mM). The increases in NF-κB activation and VCAM-1 expression caused by palmitate were markedly inhibited by co-incubation with the AMPK activator AICAR and, where studied, by expression of a constitutively active AMPK. Likewise, AMPK activation inhibited the increase in NF-κB reporter gene expression observed in HUVEC incubated with TNF-α. The results suggest that AMPK inhibits the activation of NF-κB caused by both palmitate and TNF-α. The mechanism responsible for this action, as well as its relevance to the reported anti-atherogenic actions of exercise, metformin, thiazolidinediones, and adiponectin, all of which have been shown to activate AMPK, remains to be determined.