A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain. After UV-EMS and UV-DES treatments respectively, seven mutated strains with subtle improvements in acid tolerance were obtained, and were subjected for recursive protoplast fusion. Through three rounds of genome shuffling, four shuffled strains with both higher yield and acid tolerance were obtained. The shuffled strain namely F3-21 could even survive at pH 5.2. The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here. After 48 h of shake-flask fermentation, the succinic acid concentration of F3-21 was 48% higher than that of the parent strain. When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0, the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L, which was increased by 45% compared with that of the parent strain (26.2 g/L). While pH was controlled at 6.5~7.0, the production of succinic acid in 32 h fermentation attained 40.7 g/L. When F3-21 was carried out in fed-batch fermentation, succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation. These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A. succinogenes CGMCC 1593.