Abstract: Objective  To investigate the roles of drug-resistant related gene mutation and efflux pump genes in mono-rifampicin resistance of Mycobacterium tuberculosis isolates.  Methods Twenty-three mono-rifampicin resistant clinical isolates identified in national survey of drug-resistant tuberculosis in 2007—2008 were obtained from National Tuberculosis Reference Laboratory. Direct sequencing was performed to detect the mutations in the rpoB gene. The total RNA of the rifampicin resistant strains without rpoB mutation was extracted and reverse-transcribed. The transcript level of 20 drug efflux genes was detected by real-time PCR and the RFP-related efflux pumps were screened by comparing with the corresponding results of minimum inhibitory concentration (MIC). The function of candidates in RFP-resistance was confirmed by transgenic E.coli.  Results Of 23 mono-rifampicin resistant strains, the mutations of rpoB were detected in 16 (69.6%) strains, including 9 isolates with mutation located in 531 codon, 6 isolates in 526 codon and 1 isolate in 533 codon. The isolates with Ser531Leu and His526Asp mutation displayed high-level rifampicin resistance, the MIC of which was 192 μg/ml and 256 μg/ml, respectively, while the mutations including His526Gly，His526Leu，His526Arg and Leu533Pro were related to low-level rifampicin resistance, the MIC of which was 0.75 μg/ml，4 μg/ml，2 μg/ml and 0.5 μg/ml, respectively. In the strains without rpoB mutation, the expression levels of Rv2936, Rv0783 and Rv0933 by PCR positively correlated to MIC. Moreover, the BL21 transferred with pEASY-E1-Rv2936 and pEASY-E1-Rv0783 showed higher MIC level, 32 μg/ml and 16 μg/ml, respectively, than 8 μg/ml in the control E.coli and the E.coli transferred with pEASY-E1-Rv0933.  Conclusion The different mutations in rpoB are associated with MIC level. And Rv2936 and Rv0783 may be the efflux pumps related to rifampicin resistance in Mycobacterium tuberculosis.