Abstract Secondary active transporters translocate their substrates using the electrochemical potentials of other chemicals, undergoing large-scale conformational changes. Despite extensive structural studies, the atomic details of the transport mechanism still remain elusive. Here we performed a series of all-atom molecular dynamics simulations of the triose-phosphate/phosphate translocator (TPT), which exports organic phosphates in the chloroplast stroma in strict counter exchange with inorganic phosphate (P i ). Biased sampling methods, including string method and umbrella sampling, successfully reproduced the conformational changes between the inward– and outward-facing states, along with the substrate binding. The free energy landscape of this entire TPT transition pathway demonstrated the alternating access and substrate translocation mechanisms, which revealed P i is relayed by positively charged residues along the transition pathway. Furthermore, the conserved Glu207 functions as a “molecular switch”, linking the local substrate binding and the global conformational transition. Our results provide atomic-detailed insights into the energy coupling mechanism of antiporter.