Mitochondrial ADP/ATP carriers are inhibited by two natural compounds, atractyloside (ATR) or carboxy-atractyloside (CATR), which differ by one carboxylate group. The interactions of the inhibitors with the carrier were investigated by single-molecule force spectroscopy. Transmembrane alpha helices of the ATR-inhibited carrier displayed heterogeneous mechanical and kinetic properties. Whereas alpha helix H2 showed the most brittle mechanical properties and lowest kinetic stability, alpha helix H5 was mechanically the most flexible and possessed a kinetic stability 9 orders of magnitude greater than that of alpha helix H2. In contrast, CATR-binding substantially increased the kinetic stability of alpha helix H2 and tuned the mechanical flexibility of alpha helices H5 and H6. NMR spectroscopy confirmed that the additional carboxylate group of CATR binds to the sixth alpha helix, indicating that the enhanced stability of H2 is mediated via interactions between CATR and H6.