The neurovascular unit (NVU) consists of multiple cell types including brain endothelial cells, pericytes, astrocytes, and neurons that function collectively to maintain homeostasis within the CNS microenvironment. As the principal barrier-forming component of the NVU, the endothelial cells perform an array of complex functions that require substantial energy resources. The principal metabolic pathways for producing ATP are glycolysis and mitochondrial oxidative phosphorylation. While previous studies have demonstrated that glycolysis is a primary pathway for most endothelial cells, details about the energy producing pathways of brain endothelial cells are not fully characterized. The contributions of glycolysis and mitochondrial respiration to energy metabolism are quantifiable using metabolic flux analysis that measures cellular oxygen consumption and acidification (proton production) in a closed microtiter plate format. ATP production rates are then calculated. The bioenergetics of the human brain microvascular endothelial cell line, hCMEC/D3, indicate that these cells exhibit relatively elevated rates of glycolytic flux and glycolytic ATP production, thus confirming their glycolytic nature even in the presence of abundant oxygen. Furthermore, energy producing pathways involving mitochondrial respiration are relatively low, although contributing significantly to total ATP production. Interestingly, the bioenergetics of the hCMEC/D3 cells are relatively similar to those of human primary brain microvascular endothelial cells (hBVECs). These findings allow a quantitative understanding of the bioenergetics of brain endothelial cells in a cultured and proliferative state and also provide a platform for comparative studies of disease states and conditions involving exposures to drugs or metabolic disruptors.