Abstract: Objective: To investigate the protective effects of Cordyceps militaris on antibiotic-induced intestinal damage in mice, induced by the combined administration of cefuroxime axetil and levofloxacin hydrochloride. Methods: Mice were administered high (5×), medium (3×), and low (1×) doses of cefuroxime axetil and levofloxacin hydrochloride for 14 consecutive days. The general health status of the mice, as well as changes in the morphology of the gastrointestinal tract and intestinal villi, were observed. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), creatinine (CRE), blood urea nitrogen (BUN), lipopolysaccharide (LPS), and diamine oxidase (DAO) were measured to determine the optimal antibiotic combination for inducing intestinal injury. Subsequently, the mice were then randomly divided into three groups: antibiotic injury group (MGT, 5×antibiotics), Cordyceps militaris-treated group (MGY, 5×antibiotics+0.61 g/kg.bw Cordyceps militaris), and blank group (BGT). The effects of Cordyceps militaris on the intestinal physical barrier of model mice were observed and recorded. The structure of the intestinal microbiota was analyzed using 16S rDNA high-throughput sequencing. Results: Both the medium and high doses of antibiotics caused behavioral changes in the mice, such as lethargy, hunching, and reduced food and water intake. Serum levels of DAO, ALT, AST, and LPS were significantly elevated (P<0.05). In the MG5 group (100% of individuals), gastrointestinal abnormalities such as bloating, intestinal wall transparency, villus rupture in the small intestine, submucosal hemorrhage, and edema were observed. Therefore, the high dose was determined to construct the treatment concentration for antibiotic intestinal injury. After 21 days of Cordyceps militaris treatment, the MGY group showed restored normal eating, drinking, and behavior. The morphology of the gastrointestinal tract and intestinal villi returned to normal. Serum AST levels significantly decreased to 13.56±0.82 U/L, with no significant difference compared to the BGT group (P>0.05). Compared to the MGT group, serum DAO and LPS levels in the MGY group reduced by 49.15% and 51.51% respectively (P<0.05). The intestinal microbiota richness and diversity in the MGY group showed a restorative increase. Cordyceps militaris treatment reduced the abundance of the pathogenic bacterium Klebsiella variicola in mice with intestinal injury while promoting the colonization of beneficial bacteria, including Bacteroides acidifaciens, Eubacterium_sp_14-2, Lactobacillus murinus. By the end of the experiment, no significant differences were observed between the treatment group and the BGT group (P>0.05). Conclusion: C. militaris showed significant good reparative effects against intestinal damage caused by antibiotic co-administration, indicating its potential as a functional food for development and utilization.