Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis in which a sulfur atom derived from a [2Fe-2S] ²⁺ cluster is inserted between the C6 and C9 carbons of dethiobiotin to complete the thiophane ring of biotin. The proposed reaction sequence involves reductive cleavage of S-adenosylmethionine to generate 5'-deoxyadenosyl radicals, which then abstract hydrogen atoms from dethiobiotin. The putative dethiobiotinyl radical intermediates are then quenched through the stepwise addition of the bridging sulfide of a protein-derived [2Fe-2S]²⁺ cluster to generate biotin. This results in destruction of the [2Fe-2S] ²⁺ cluster rendering BioB inactive after only one turnover. The present work demonstrates that the loss of this cluster is associated with a decrease in thermal stability and an increase in proteolytic susceptibility, both in vitro and in vivo. Facile unfolding of the apoprotein following turnover may be an evolved feature of biotin synthase that facilitates repair of the [2Fe-2S]²⁺ cluster and allows multiple turnovers of the enzyme in vivo. In order for BioB to regain its enzymatic function, the [2Fe-2S]²⁺ cluster of BioB needs to be regenerated, a process that is catalyzed in vivo by the Iron- Sulfur Cluster assembly (ISC) system. Presented here is the discovery of a complex between BioB, in multiple [Fe-S] cluster states, and HscA, an Hsp70 class molecular chaperone encoded within the ISC system. We show, using a MW filtration assay, that HscA binds with increasing affinity as BioB loses its iron-sulfur clusters. HscA binds BioB in a hydrophobic, ATP/ADP-independent manner in which two HscA molecules bind one dimer of BioB. We also report that HscA recognizes a loop on BioB located at the bottom of its (αβ)₈ barrel near the [2Fe-2S] cluster. An interaction between BioB and HscA suggests this chaperone may play a role in the maintenance of BioB after turnover and/or targeting of [Fe-S] cluster machinery to the apoprotein substrate.