Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of L. agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4 hours of treatment at pH 2, and under the condition of 0.5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77.8%, and exhibits 67.1% auto-aggregation and 63.2% aggregation with Enterotoxigenic Escherichia coli 10 (ETEC 10). FITC fluorescence labeling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P < 0.05), but no significant difference from ileum. L. agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14.2 mm and 15.4 mm, respectively. L. agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhea pathogen infection.This article is protected by copyright. All rights reserved.