Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme of the Calvin reductive pentose phosphate cycle. Two sets of structural genes (cbbLS-1 and -2) for form I RubisCO have been previously identified in the Mycobacterium sp. strain JC1, which is able to grow on carbon monoxide (CO) or methanol as sole sources of carbon and energy. Northern blot and reverse transcriptase PCR showed that the cbbLS-1 and -2 genes are expressed in cells grown on either carbon monoxide (CO) or methanol, but not in cells grown in nutrient broth. A promoter assay revealed that the cbbLS-2 promoter has a higher activity than the cbbLS-1 promoter in both CO- and methanol-grown cells, and that the activities of both promoters were higher in CO-grown cells than in methanol-grown cells. A gel mobility shift assay and footprinting assays showed that CbbR expressed in Escherichia coli from a cbbR gene, which is located downstream of cbbLS-1 and transcribed in the same orientation as that of the cbbLS genes, specifically bound to the promoter regions of the cbbLS-1 and -2 genes containing inverted repeat sequence. A DNase I footprinting assay revealed that CbbR protected positions -59 to -3 and -119 to -78 of the cbbLS-1 and -2 promoters, respectively. Overexpression of CbbR induced the transcription of RubisCO genes in Mycobacterium sp. strain JC1 grown in nutrient broth. Our results suggest that the CbbR product from a single cbbR gene may positively regulate two cbbLS operons in the Mycobacterium sp. strain JC1 as is the case for Rhodobacter sphaeroides and Cupriavidus necator.