Significance: Biogenesis of iron–sulfur proteins is a complex process requiring a large number of accessory proteins. In eukaryotes, [2Fe-2S] clusters are synthesized in mitochondria on a scaffold protein. The cluster is then released to monothiol glutaredoxin 5 (GRX5), which was proposed to mediate the transfer of [2Fe-2S] clusters from the scaffold protein to several target proteins, but its precise molecular function remains to be clarified. By investigating the molecular recognition between human GRX5 and its partner proteins (human ISCA1 and ISCA2) and characterizing at the molecular level the cluster transfer process between them, we have shown that a switch between two conformational states of holo GRX5 drives the cluster transfer event, which occurs by a specific protein–protein recognition process.