Genome shuffling for improving the activity of alkaline pectinase in Bacillus subtilis FS105 and its molecular mechanism were investigated. The fused strain B. subtilis FS105 with the highest activity of alkaline pectinase was obtained after two rounds of genome shuffling. The activity of alkaline pectinase in B. subtilis FS105 was 499 U/ml, which was improved by 1.6 times compared to that in original strain. To elucidate its molecular mechanism, rpsL gene sequences from original and fused strains were cloned and aligned, and the space structure of their coding proteins were also analyzed and compared. The alignment of the rpsL gene sequences indicated that three bases G, G and C were respectively replaced by A, A and G in the positions 52, 408 and 409 after genome shuffling. This resulted in the substitution of two amino acid residues in ribosomal protein S12: D18N and P137A, and therefore improving the biosynthesis of alkaline pectinase. This study lays a foundation for improving the activity of alkaline pectinase by genome shuffling and understanding its molecular mechanism.