CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.