Objective To construct the prokaryotic expression vector carrying Mycobacterium tuberculosis gene mmpL6 (rv1557), overexpress and purify the recombinant MmpL6 (Rv1557) protein in Escherichia coli (E. coli).Methods The DNA fragment of MmpL6 was amplified by polymerase chain reaction using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template. The target gene was cloned into pET21b vector to construct the pET21b-MmpL6 expression plasmid, and then transformed into E. coli for protein expression. Recombinant protein expression was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and detected by SDS-PAGE combined with Western blot. The overexpressed MmpL6 protein was purified by Ni-affinity chromatography and gel filtration chromatography. Results The recombinant pET21b-MmpL6 plasmid was successfully constructed and the highest expression level was obtained at 25 ℃ in Rosetta strain induced by IPTG. Conclusion The successful expression and purification of MmpL6 in E. coli lays the foundation for the further structure and function studies of the protein, and also provides clues to the design of anti-tuberculosis drugs targeting efflux pump proteins.