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Aims: To detect the effect of pepsin-hydrolysate conglycinin (PTC) on the growth of Escherichia coli O138in vitro, and investigate the effect of PTC on intestinal microbial community of mice after E. coli infection. Methods and Results: Serial dilution method was used to detect the antibacterial activity of PTC in 96-well cell-cultivated plates. Fifty-five KM mice were randomly assigned to five groups: normal, feeding-E. coli control, HCl-full hydrolysis of conglycinin, conglycinin and PTC. Orally administrated with hydrolysates from conglycinin for 21 days, each mouse was fed with 2 × 108 CFU ml−1 of E. coli O138 on the 22nd day. The mice activities were monitored and polymerase chain reaction-denaturing gradient gel electrophoresis was used to analyse the microbial community in mice faeces. The results showed that PTC could inhibit growth of E. coli O138 at nitrogen concentrations of more than 520 mg l−1. There was high similarity of intestinal microbial community in mice between PTC and normal groups. Conclusion: PTC inhibits growth of E. coli O138, keeps mice healthy following oral administration of E. coli infection and maintains a balanced active microbial community in their gastrointestinal tract. Significance and Impact of the Study: This study demonstrated the antibacterial activity of PTC against E. coli and its ability to maintain healthy intestinal microbial community in mice even after they were infected with E. coli. This observation is significant in the application of PTC to prevent gastrointestinal diseases caused by E. coli and unbalanced intestinal microflora.