During infection of macrophages, Mycobacterium tuberculosis, the pathogen that causes tuberculosis, utilizes fatty acids as a major carbon source. However, little is known about the coordination of the central carbon metabolism of M. tuberculosis with its chromosomal replication, particularly during infection. A recently characterized transcription factor called PrpR is known to directly regulate the genes involved in fatty acid catabolism by M. tuberculosis. Here, we report for the first time that PrpR also regulates the dnaA gene, which encodes the DnaA initiator protein responsible for initiating chromosomal replication. Using cell-free systems and intact cells, we demonstrated an interaction between PrpR and the dnaA promoter region. Moreover, real-time quantitative reverse-transcription PCR analysis revealed that PrpR acts as a transcriptional repressor of dnaA when propionate (a product of odd-chain-length fatty acid catabolism) was used as the sole carbon source. We hypothesize that PrpR may be an important element of the complex regulatory system(s) required for tubercle bacilli to survive within macrophages, presumably coordinating the catabolism of host-derived fatty acids with chromosomal replication.