Uncoupling protein-2 (UCP2) belongs to the mitochondrial carrier family and has been thought to be involved in suppressing mitochondrial ROS production through uncoupling mitochondrial respiration from ATP synthesis. However, we show here that loss of function of UCP2 does not result in a significant increase in ROS production or an increased propensity for cells to undergo senescence in culture. Instead, Ucp2-/- cells display enhanced proliferation associated with a metabolic switch from fatty acid oxidation to glucose metabolism. This metabolic switch requires the unrestricted availability of glucose, and Ucp2-/- cells more readily activate autophagy than wild-type cells when deprived of glucose. Altogether, these results suggest that UCP2 promotes mitochondrial fatty acid oxidation while limiting mitochondrial catabolism of pyruvate. The persistence of fatty acid catabolism in Ucp2+/+ cells during a proliferative response correlates with reduced cell proliferation and enhances resistance to glucose starvation-induced autophagy.—Pecqueur, C., Bui, T., Gelly, C., Hauchard, J., Barbot, C., Bouillaud, F., Ricquier, D., Miroux, B., Thompson, C. B. Uncoupling protein-2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization.