The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types. Display omitted •RESPYR is a genetically encoded biosensor based on bioluminescence energy transfer•RESPYR enables monitoring mitochondrial pyruvate carrier activity in real time•RESPYR enables analysis of the mitochondrial pyruvate carrier in single cells•RESPYR provides a non-invasive technology to monitor cellular energy metabolism Current techniques for monitoring influx of pyruvate into mitochondria provide only an indirect measure of mitochondrial pryuvate carrier (MPC) activity. Compan et al. have engineered a biosensor that enables monitoring MPC activity in real time to investigate metabolism in living cells.