Fe–S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe–S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe–S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe–S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe–S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe–S cluster biogenesis.