10xHis-RR-MPI was designed to shorten the process of producing recombinant human insulin for treatment of diabetes in Vietnam. Trypsin cleavage sites (RR) were inserted between 10xHis tag and MPI, thus 10xHis tag and mini-peptide C were removed from refolded 10xHis-RR-MPI in one step under the catalysis of trypsin protease. Gene 10xHis-RR-MPI was amplified by PCR, cloned into pET -43.la(+) to construct recombinant plasmid pHTI expressing 10xHis-RR-MPI under the control of T7 promoter. E. coli BL21(DE3)/pHTI strain was established by chemical transformation of pHTI into E. coli BL21(DE3), shakily cultivated in LB medium, added IPTG to induce the T7 promoter. 10xHis-RR-MPI was over-expressed and accumulated as inclusion bodies in cytoplasm and occured about 14 percent of total protein after 10h induction when analyzing by SDS-PAGE, Western blot, and Quantity-One software. 10xHis-RR-MPI was purified through a one-step Ni-NTA affinity column under denaturing conditions. 18.9 mg of purified 10xHis-RR-MPI was obtained from 500 ml LB medium of shakily cultured recombinant bacteria when quantifying protein using Bradford method.