Mitochondrial ATP-Mg/Pi carriers import adenine nucleotides into the mitochondrial matrix and export phosphate to the cytosol. They are calcium-regulated to control the size of the matrix adenine nucleotide pool in response to cellular energetic demands. They consist of three domains: an N-terminal regulatory domain containing four calcium-binding EF-hands， a linker loop domain with an amphipathic α-helix and a C-terminal mitochondrial carrier domain for the transport of substrates. Here， we use thermostability assays to demonstrate that the carrier is regulated by calcium via a locking pin mechanism involving the amphipathic α-helix. When calcium levels in the intermembrane space are high， the N-terminus of the amphipathic α-helix is bound to a cleft in the regulatory domain， leading to substrate transport by the carrier domain. When calcium levels drop， the cleft closes， and the amphipathic α-helix is released to bind to the carrier domain via its C-terminus， locking the carrier in an inhibited state.