Abstract: Bacillus subtilis nja A1B2-2 as the starting strain, based on the optimum conditions for protoplast formation, regeneration and protoplasts fusion, genome shuffling was carried out between four mutant strains screened from phys-chem mutagenesis. The results showed when the hypertonic washing solution 0.6mol/L Na Cl, 0.1mg/m L lysozyme treating for 40 min, spreading on gregeneration medium NB with hypertonic SMM, protoplast formation rate and the regeneration rate respectively reached 95.49% and 89.25%. After optimizing protoplast fusion conditions, its rate was up to 1.39 ×10-3. Then two round of genome shuffling was launched between four high-yield Subtilosin A strains. With the selection system of double inactivation of parental protoplasts, a stable and high-yield strain R2-264 was selected out with the maximum production of Subtilosin A at 17.59mg/L, which was 3.58 times higher than the starting strain.