Cellular senescence is now recognized as an important mechanism of tumor suppression, and the accumulation of senescent cells may contribute to the aging of various human tissues. Alterations of the cellular energy metabolism are considered key events in tumorigenesis and are also known to play an important role for aging processes in lower eukaryotic model systems. In this study, we addressed senescence-associated changes in the energy metabolism of human endothelial cells, using the HUVEC model of in vitro senescence. We observed a drastic reduction in cellular ATP levels in senescent endothelial cells. Although consumption of glucose and production of lactate significantly increased in senescent cells, no correlation was found between both metabolite conversion rates, neither in young endothelial cells nor in the senescent cells, which indicates that glycolysis is not the main energy source in HUVEC. On the other hand, glutamine consumption was increased in senescent HUVEC and inhibition of glutaminolysis by DON, a specific inhibitor of glutaminase, led to a significant reduction in the proliferative capacity of both early passage and late passage cells. Moreover, inhibition of glutaminase activity induced a senescent-like phenotype in young HUVEC within two passages. Together, the data indicate that glutaminolysis is an important energy source in endothelial cells and that alterations in this pathway play a role in endothelial cell senescence.