Abstract:［Objective］Reactive oxygen species are natural products of metabolism in aerobic organisms，which lead to oxidative damage，such as DNA mutation，protein inactivation and drug resistance．MSMEG_3312 was predicted as a hemerythrin-like protein，which can carry oxygen and reversibly bind to oxygen，thus it might play important roles in the process of oxygen metabolism．In this study，we explored the role of MSMEG_3312 in drug resistance.［Methods］On the basis of bioinformatics，we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical α-helix at secondary structure．To explore potential functions of MSMEG_3312，we constructed the msmeg_3312 knockout strain and compare the susceptibility to various drugs to its parent strain，mc2155．In addition，we also measured the promoter response when treatment of erythromycin．［Results］Genetic results showed that MSMEG_3312 is not necessary for M. smegmatis growth at 7H9 rich medium．The msmeg _ 3312 knockout strain showed increased erythromycin resistance． Moreover，the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis．However，there were no different MICs of other antibiotics，targets for protein synthesis inhibition，but not 50S subunit，such as tetracyclines，aminoglycosides and chloramphenicol．Moreover，we also showed that the promoter of msmeg_3312 responses to erythromycin． ［Conclusions］Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.