The zoonotic enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can disrupt intestinal epithelial barrier function and in turn leading to serious intestinal and systemic disease. PR39 could effectively inhibit the growth of Gram-negative bacteria, but there is little knowledge of its effects on intestinal barrier function and the microbiota in E. coli-challenged mice. In this study, an intestinal disease caused by EHEC O157:H7 was established, to analyze the effect of PR39 on EHEC O157:H7 induced intestinal epithelial barrier injury and disorder. Interestingly, PR39 attenuated EHEC O157:H7-induced systemic symptoms and significantly decreased mortality and the degree of E. coli shedding in faeces. Furthermore, the infiltration index of macrophages and neutrophils in intestine of the PR39 treatment group were obviously attenuated, along with the level of apoptosis. PR39 treatment group had distinctly improved tight junction associated proteins' expression after EHEC O157:H7 caused injury. Additionally, the sequencing analysis of cecum microbiota showed that PR39 altered the abnormal increase in Bacteroides caused by EHEC O157:H7 and promoted the growth of probiotics such as Lactobacillus. In conclusion, cathelicidin-derived PR39 could effectively improve EHEC O157:H7-induced epithelial barrier injury, and dysfunction of immune and microbiota homeostasis in the intestinal tract, indicating that PR39 could be an excellent potential drug for zoonotic EHEC O157:H7-related intestinal disease.