The B27K-DTrI insulin (human insulin with B28–30 removed and B27 Thr replaced by Lys) was reported to have superior monomeric property with 80% insulin activity in vivo. It has potential use as a new fast-acting analog of insulin. We cloned the monomeric insulin B27 DTrI precursor (MIP) into the pTWIN1 vector, and prepared by intein mediated expression in Escherichia coli. After tryptic digestion, the MIP was converted to B27K-DTrI insulin. The product was purified by HPLC. The mass spectrometry showed that the molecular mass of purified B27K-DTrI was consistent with the theoretical value.Highlights► The monomeric insulin B27 DTrI precursor (MIP) was prepared by intein mediated expression in Escherichia coli. ► The B27K-DTrI insulin was obtained by tryptic digestion, further purified by HPLC and analyzed by mass spectrometry. ► The molecular mass of purified B27K-DTrI was consistent with the theoretical value.