Rhodobacter sphaeroides is a non-sulfur photosynthetic bacterium that possesses two cbb operons, cbb (I) and cbb (II) , encoding enzymes involved in the Calvin-Bensom-Bassham reductive pentose phosphate pathway of carbon dioxide fixation. In the present study, a number of molecules have been identified that have the ability to alter the in vivo DNA-binding properties of CbbR protein in R. sphaeroides. The CbbR-binding sites on the cbb operon in R. sphaeroides were characterized by chromatin immunoprecipitation (ChIP) assay. The ChIP assay indicated that the CbbR protein binds specifically to the upstream regions cbbF in cbb (I) operon and cfxB in cbb (II) operon. The change in the binding of CbbR to the upstream of cbbF and cfxB in the presence of RuBP, fructose 1,6-bisphosphate, NADPH, KH2PO4 was observed under anaerobic, aerobic, aerobic light-dark, and aerobic dark conditions, respectively. From these results, the role of different co-inducer molecules in influencing the interactions of CbbR with the binding site within cbb operon has been ascertained. The biosynthetic intermediates and other potential metabolic effectors have been observed to play an important role in the regulatory mechanism.