Limosilactobacillus reuteri is a vertebrate symbiont that is widely appreciated as being of significant ecological importance for human health. As a unique feature, L. reuteri converts glycerol to the antimicrobial compound reuterin using enzymes encoded in its propanediol-utilization operon and evolves with host-driven diversification. Reuterin-producing L. reuteri HLRE13 was selectively isolated from poultry previously and confirmed to inhibit the growth of Staphylococcus aureus in vitro. However, it remains unclear whether L. reuteri HLRE13 retains these antagonistic properties when ingested in specific-pathogen-free mice. Here, we investigated the ameliorative effects and potential mechanisms of action of L. reuteri HLRE13 in combination with glycerol on S. aureus-induced infection phenotypes in mice. Firstly, our results confirmed that L. reuteri HLRE13 effectively inhibited the intestinal colonization of S. aureus CMCC26003; Secondly, L. reuteri HLRE13 combined with glycerol could alleviate the intestinal tissues damage caused by S. aureus through increasing the expression of ZO-1, Occludin, and MUC-2, ameliorate the intestinal systemic inflammatory response, and maintain the balance of gut microbiota by increasing the relative abundance of Lactobacillus and reducing the relative abundance of Staphylococcus. Furthermore, the colonization resistance was also found on L. reuteri HLRE13 combined with glycerol against S. aureus in pseudo germ-free mice, and they exerted the similar effects on alleviating intestinal damage and improving immune function. Combining these results, we speculate that reuterin-producing L. reuteri antagonize S. aureus in mice without the gut microbiota-dependent manner. Overall, our findings will provide a theoretical foundation for the scientific cognition of L. reiteri in maintaining intestinal health by producing reuterin.