Acidithiobacillus ferrooxidans is an obligately chemolithoautotrophic, γ-proteobacterium that fixes CO2 by the Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle. Our objective is to identify genes potentially involved in CO2 fixation and to advance our understanding of how they might be regulated in response to environmental signals. Bioinformatic analyses, based on the complete genome sequence of the type strain ATCC 23270, identified five cbb gene clusters four of which we show experimentally to be operons. These operons are predicted to encode: (i) the components of the carboxysome and one copy of form I RubisCO (cbb1 operon), (ii) a second copy of form I RubisCO (cbb2 operon), (iii) enzymes of central carbon metabolism (cbb3 operon), (iv) a phosphoribulokinase and enzymes of sulfur metabolism (cbb4 operon) and RubisCO form II (cbb5 gene cluster). In addition, the gene for a LysR-type transcriptional regulator CbbR was identified immediately upstream and in divergent orientation to the cbb1 operon and another associated with the cbb5 gene cluster. A. ferrooxidans was grown under different concentrations of CO2 (2.5 to 20% [v/v]), and levels of mRNA and protein were evaluated by qPCR and Western blotting, respectively. CbbR binding to predicted promoter regions of operons cbb1-4 was assayed by EMSA This information permitted the formulation of models explaining how these operons might be regulated by environmental CO2 concentrations. These models were evaluated in vivo in a heterologous host, using cloned A. ferrooxidans cbbR to complement a mutant of the facultative chemoautotroph Ralstonia eutropha H16 lacking a functional cbbR. Cloned copies of A. ferrooxidans promoter regions were also introduced into R. eutropha to evaluate their ability to drive reporter gene expression. This work lays the framework for further studies that should result in a more comprehensive picture of how CO2 fixation is regulated in A. ferrooxidans.