Simple SummaryThe interaction between intestinal microbiota and host plays a key role in the development of intestinal diseases. Salmonella enteritidis infection is a common cause of human gastroenteritis, which can lead to gastrointestinal dysfunction and microecological disorder. It is an important public health problem all over the world. Lactic acid bacteria are among the main probiotics in the intestinal microbiota, which is one of the main defense lines against intestinal pathogens. It is now also considered to be an effective alternative antibiotic to fight pathogenic bacterial infections. In order to explore the potential mechanism of lactic acid bacteria against S. enteritidis infection, this study started from the perspective of lactic acid bacteria regulating the intestinal microbiota and the infection of antigenic bacteria. The probiotic strain Lactobacillus reuteri S5 (L. reuteri S5) was used to establish an animal model of S. enteritidis infection in broilers. The regulatory effect of L. reuteri S5 on the intestinal microbiota structure of chickens infected with S. enteritidis is studied by high throughput sequencing technology. The results showed that L. reuteri S5 could regulate the composition and abundance of intestinal microbiota and resist the infection of S. enteritidis.To understand the mechanism of lactic acid bacteria against Salmonella enteritidis infection; we examined how lactic acid bacteria regulated the intestinal microbiota to resist infection by pathogenic bacteria. The probiotic strain Lactobacillus reuteri S5 was used to construct an animal model of S. enteritidis infected broilers. A high-throughput sequencing technology was used to analyze the regulatory effects of L. reuteri S5 on the structure of the intestinal microbiota of broilers infected with S. enteritidis; and to examine the possible defense mechanism they used. Our results showed that the administration of L. reuteri S5 reduced colonization of S. enteritidis (p < 0.05), decreased intestinal permeability (p < 0.05), and reduced the bacterial displacement likely due by S. enteritidis colonization (p < 0.05), suggesting some enhancement of the intestinal barrier function. Furthermore, L. reuteri S5 increased the number of operational taxonomic units (OTUs) in the chicken cecal microflora and the relative abundance of Lactobacillaceae and decreased the relative abundance of Enterobacteriaceae. These results suggest that the lactic acid bacterium L. reuteri S5 protected the intestinal microbiota of chickens against S. enteritidis infection.