The genetic aspects of DnaA mediated initiation of oriC replication in mycobacteria are largely unknown. To get insights into the replication initiation process in mycobacteria, we characterized Mycobacterium tuberculosis DnaA and its interactions with oriC. We show that the replacement of Mycobacterium smegmatis dnaA with the M. tuberculosis counterpart expressed from its native promoter resulted in temperature-sensitive (TS) phenotype. However, the TS phenotype was abolished when the M. tuberculosis dnaA was expressed from the inducible amidase promoter, which produces elevated levels of DnaA. We provide evidence that M. tuberculosis dnaA promoter activity was unaffected at non-permissive temperature, but the DnaA protein was found to be unstable indicating that protein factors stabilizing M. tuberculosis DnaA are absent in M. smegmatis. Finally, we show by surface plasmon resonance that the M. tuberculosis DnaA interacts with M. smegmatis oriC, similar to its cognate oriC indicating that the binding interactions between in vitro folded DnaA and oriC are unaffected. Our results suggest that Mtb DnaA functions as a partially active protein in M. smegmatis, hence is not as proficient as M. smegmatis counterpart in optimally driving the M. smegmatis oriC replication machinery.