Extracellular tumor acidosis largely results from an exacerbated glycolytic flux in cancer and cancerassociated cells. Conversely, little is known about how tumor cells adapt their metabolism to acidosis. Here, we demonstrate that long-term exposure of cancer cells to acidic pH leads to a metabolic reprogramming toward glutamine metabolism. This switch is triggered by the need to reduce the production of protons from glycolysis and further maintained by the NAD þ -dependent increase in SIRT1 deacetylase activity to ensure intracellular pH homeostasis. A consecutive increase in HIF2a activity promotes the expression of various transporters and enzymes supporting the reductive and oxidative glutamine metabolism, whereas a reduction in functional HIF1a expression consolidates the inhibition of glycolysis. Finally, in vitro and in vivo experiments document that acidosis accounts for a net increase in tumor sensitivity to inhibitors of SIRT1 and glutaminase GLS1. These findings highlight the influence that tumor acidosis and metabolism exert on each other.