Irinotecan (CPT-11) is a chemotherapy agent commonly used for the treatment of gastrointestinal tumors, with diarrhea being a frequent adverse effect. Hesperidin is a flavonoid abundant in citrus fruits and has shown potential in managing CPT-11-induced diarrhea (CID). However, the mechanisms underlying its effects remain unclear. This study established a mouse model of CID using CPT-11 administration to evaluate the effects of hesperidin on diarrhea severity, intestinal pathology, gut microbiota composition, and metabolite profiles by conducting biochemical analysis, histopathology, immunohistochemistry, 16S rRNA sequencing, and untargeted metabolomics. In addition, transcriptomic analysis, molecular docking, and molecular dynamics simulations were conducted to investigate potential mechanisms of action. Hesperidin supplementation was found to significantly alleviate CID in mice. Analysis of gut microbiota using 16S rRNA sequencing revealed that hesperidin improved microbial composition, with key taxa such as