Acidithiobacillus ferrooxidans, an important microorganism in bioleaching industry, has been sequenced recently, and from the annotated information, there are four genes involved in copper homeostasis. Sequence analysis showed that two of them, Afe0329 and Afe0663, were high homologous (94.43% identity). With the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) cloning approach, the differential gene expression of these two high homologous genes in a genome was successfully identified for the first time. In comparison with Afe0663, Afe0329 was highly expressed grown in the medium with copper, and the restriction fragment length polymorphism (RFLP) profile showed that 96% of lanes were products of Afe0329. Analysis of the protein sequence encoded by Afe0329 suggested a conserved domain of P1b3-type ATPase, which is a heavy-metal pump, and, to be unexpected, the molecular modeling revealed that the amino acids determining the type of heavy-metal pumps were responsible for the gate of the copper ion channel in the transmembrane area of the protein. The activity of P1b-type ATPase disrupted in Escherichia coli could be partially rescued by complementation by the plasmid-carrying Afe0329 gene. All of these results suggest that a copper homeostasis mechanism including P-type ATPase is of importance for the survival of this extremophilic microorganism.