Plant type [2Fe-2S] ferredoxins function primarily as Reversible unfolding and electron transfer proteins in photosynthesis. Studying the unfolding folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding- refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro.