The activity of many membrane proteins depends markedly on the pH. Pinpointing the amino acids forming the pH sensor domains of these proteins remains challenging for current experimental techniques. Combining molecular dynamics simulations and pKa predictions with in vitro transport assays, we have revealed the molecular basis of the pH dependence of the mitochondrial carrier mediating the exchanges of ADP3– and ATP4– across the inner mitochondrial membrane. We have demonstrated that the transport activity of this mitochondrial carrier depends on the protonation state of both the substrate and a unique, highly conserved residue of the protein. The original strategy proposed here offers a convenient framework for identifying pH-sensitive residues in membrane proteins in such cases where one single amino acid is involved. Our findings are envisioned to help toward the rational design of active compounds ranging from drugs to biosensors.