Abstract: In order to prepare a cyclic polypeptide cLunasin by the method of total biosynthesis, the linear precursor aLunasin was designed for cLunasin synthesis, and a fusion expression vector with the double tags of MBP and SUMO was constructed. After the recombinant expression of aLunasin by Escherichia coli, the cLunasin was catalyzed to synthesize by the polypeptide ligase Butelase 1. The effects of cyclization on the thermal stability, structural properties and anticolorectal cancer activity of the peptide were further investigated by circular dichroism spectroscopy, endogenous fluorescence spectroscopy and cell experiments. The recombinant protein was successfully expressed after induction at 37 ℃, and aLunasin was successfully cyclized by Butelase 1, which was identified by the mass spectrometry identification. And 6.36 mg of cLunasin could be prepared form 1 liter of fermentation broth by the total biosynthesis. Cyclization could get the peptide structure more ordered and more compact, indicated by circular dichroism spectroscopy and endogenous fluorescence spectroscopy. Morover, the thermal stability of peptide was improved after cyclization. After incubation at 97 ℃ for 30 min, the peptide retention for cLunasin increased to 74.9%, while that of Lunasin was 62.3%. The half maximal inhibitory concentration of cLunasin on colorectal cancer HCT-116 cells was determined to 15.2 μmol/L, which was lower than that of for Lunasin 30.3 μmol/L, indicating that the anticolorectal cancer activity was enhanced significantly by cyclization. A green biosynthesis method for cLunasin was established, and an useful exploring was provided for the modification and recombinant expression of active peptides.