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The melibiose permease MelB is a well-studied Na⁺-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium. In the absence of substrate, MelB equally populates two different states, from which one shows higher mechanical structural stability with additional stabilization of the cytoplasmic middle-loop C3. In the presence of either melibiose or a coupling Na⁺-cation, however, MelB increasingly populates the mechanically less stable state, which shows a destabilized middle-loop C3. In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant. Our findings describe how both substrates guide MelB transporters to …