An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe2S2] cluster could be assembled in apoIscU with Fe2+ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize L-cysteine and Fe2+ to synthesize [Fe2S2] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe2S2] cluster.