Background: Endothelial cells form a highly dynamic barrier between blood and tissues and can be activated in response to angiogenic and inflammatory stimuli. Endothelial cells rely on glycolysis for ATP production, and it was recently demonstrated that inhibition of endothelial glycolysis attenuates angiogenic activation. We wanted to examine the metabolic profile of inflammatory activated endothelial cells and explore the role of glycolysis in endothelial inflammatory activation. Methods: Human umbilical vein endothelial cells were stimulated with IL-1β and the rates of glucose uptake, glycolysis and fatty acid oxidation were determined using radioactively labelled substrates. The expression of the metabolic enzymes PFKFB3 and CPT1A were determined by qPCR and confirmed on the protein level by immunoblotting. Glycolysis was partially inhibited using 3PO, a small molecule inhibitor of PFKFB3, and IL-1β induced adhesion molecule expression was assessed using cell ELISA and qPCR. Activation of NFκB was assessed using a NFκB luciferase assay kit and immunoblotting against phosphorylated IκBα, IKKα/β and JNK following stimulation with IL-1β and TNF. The levels of pan-O-GlcNAcylation in whole cell lysates were determined by immunoblotting. Results: The endothelial glucose uptake was increased by approximately 60% after stimulation with IL-1β (P<0.001), correlating with a steady increase in the rate of glycolysis and a decrease in fatty acid oxidation. The increase of glycolysis was associated with increased expression of the glycolytic enzyme PFKFB3, while CPT1A, the rate limiting enzyme of fatty acid oxidation was not significantly affected by inflammatory activation. Inhibition of glycolysis attenuated endothelial upregulation of the adhesion molecules E-selectin and VCAM-1, correlating with reduced phosphorylation of key signalling molecules in both the NFκB and the JNK pathways. Conclusion: We have shown that endothelial cells upregulate their rate of glycolysis in response to IL-1β stimulation and that this correlates with an increased expression of the rate limiting glycolytic enzyme PFKFB3. Pharmacological inhibition of glycolysis reduced the upregulation of adhesion molecules involved in in leukocyte recruitment by attenuating activation of inflammatory signalling cascades, raising the question whether PFKFB3 could be a potential therapeutic target in inflammatory disease. The exact molecular mechanisms by which 3PO inhibits phosphorylation of signalling molecules has not yet been determined and will be targeted in future experiments.