The Iro protein was proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, it is a member of HiPIP family with the iron-sulfur cluster for electron transfer. The gene of Iro protein from A. ferrooxidans Fe-1 was cloned and then successfully expressed in Escherichia coli, finally purified by one-step affinity chromatography to homogeneity. The recombinant protein was observed to be dimer. The molecular mass of a monomer containing the [Fe4S4] cluster was 6847.35 Da by MALDI-TOF-MS. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Molecular modelling for the protein revealed that Cys20, Cys23, Cys32 and Cys45 were in ligation with the iron-sulfur cluster, and Tyr10 was important for the stability of the [Fe4S4] cluster. As we know, this is the first report of expression in E. coli of the Iro protein from A. ferrooxidans Fe-1.