Abstract: An agar diffusion method was used to investigate the antimicrobial activities of fermentation broth of Lactobacillus acidophilus(L. acidophilus), and Clostridium butyricum(C. butyricum)against Shigella flexneri(S. flexneri)infection in vitro. It was found that cell-free culture supernatants(CFCSs)of L. acidophilus and C. butyricum possessed remarkable synergistic anti-S. flexneri activity in vitro and the antimicrobial activity of the mixed culture was 17. 2% and 22. 4% greater, than single strains alone, respectively. Meanwhile, there was a symbiotic relationship between L. acidophilus and C. butyricum. The result showed that the biomass accumulation of the mixed culture reached 4. 27 g/L(DCW)and increased by 6. 0% and 30. 6% compared to the L. acidophilus and C. butyricum, respectively. Moreover, L. acidophilus and C. butyricum clearly inhibited S. flexneri adhesion to Caco-2 cells by 75. 8% and 81. 2%, and the combination of these two probiotic strains demonstrated the highest inhibition rate, reaching 84. 2%, while the viability of Caco-2 cells treated with L. acidophilus, C. butyricum or their combination was increased by 11. 5%, 12. 5% and 22. 9%, respectively. The synergistic effect of L. acidophilus and C. butyricum provided better protection against Shigella flexneri infection, which represents a promising alternative therapy for shigellosis.