Objective To explore the structure and functions of Rv0037c protein, a member of major facilitator superfamily (MFS) proteins of Mycobacterium tuberculosis. Methods Bioinformatics analysis on Rv0037c protein was performed with TMHMM Server v.2.0, Swiss-model and DNAMAN software. The PMV261-Rv0037c overexpressed strain was constructed in Mycobacterium smegmatis, pMV261/Msm control strain was obtained, its biolo-gical function was analyzed. Results Rv0037c protein contained 11 transmembrance domains, the C/N domain consisted of "3+2" helical bundles, which was highly conservative in genus Mycobacterium. The growth kinetics curves of the overexpressed strain and the control strain almost completely overlapped, and there was no statistical difference in growth between the overexpressed strain and the control strain (P>0.05). The OD₆₀₀ of the control strain was 2.567±0.162, while that of the overexpressed strain was 2.419±0.456, there was no statistical diffe-rence in biofilm growth between the two strains (P>0.05). The accumulation of ethidium bromide (EB) in overexpressed strain was lower than that in control strain, difference was statistically significant (P<0.001). There was no statistically significant difference in the minimum inhibitory concentration (MIC) of two bacterial strains against various drugs (both P>0.05). Conclusion The Rv0037c protein is a special member of the Mycobacterium MFS family proteins and has a very conserved structure. Overexpression of Rv0037c has no effect on the growth, colony morphology, sliding ability, biofilm formation, and MIC of Mycobacterium smegmatis, but can significantly reduce the accumulation of EB.