Rifampin (RIF) susceptibility is a key factor in determining the treatment effectiveness of the standardized treatment regimens. In Mycobacterium tuberculosis, both target gene mutation and the efflux pump play major roles in the resistance to antituberculosis drugs. By eliminating RIF-resistant strains with rpoB mutation, the choice of RIF-monoresistant strains may allow us to identify the RIF-specific efflux pump genes. This study explored the RIF monoresistance mechanism in M. tuberculosis. Data from DNA sequencing and MIC measurements revealed that specific mutations, including Ser531Leu and His526Asp in RpoB, show high-level drug resistance. Three-dimensional structure modeling provided further evidence that the affinity between RIF and RpoB mutants was in accordance with the drug resistance level of the corresponding isolates. Furthermore, transcription-level analysis among the nonmutated isolates indicated that three efflux pumps (Rv0783, Rv2936, and Rv0933) might be involved in exporting RIF from the cell. Compared to 8 μg/ml for wild-type Escherichia coli, the MICs for the transgenic E. coli strains with either Rv0783 or Rv2936 were 32 and 16 μg/ml, respectively. In conclusion, our study indicated that several RpoB mutant types, including Ser531Leu and His526Asp, show high-level RIF resistance attributed to low affinity between RpoB mutant proteins and RIF. In addition, this work demonstrates that Rv2936 and Rv0783 may be responsible for low-level resistance to RIF by exporting RIF from cells. The predicted structure of RpoB and the newly identified efflux pumps in this study will provide a novel approach to design new drugs and develop novel diagnosis technologies.