Ganoderma lucidum sporoderm-broken spore powder (GLSP) has been historically documented for its anti-aging and anti-tumor effects. Although several studies have reported that GLSP and related substance may improve physiological function by regulating the gut microbiota, the underlying mechanisms and their effect on intestinal function remains largely unknown. In this study, we comprehensively investigated the impact of dietary GLSP on intestinal epithelial dynamics and barrier function, assessed by single-cell RNA sequencing (scRNA-seq), using murine models spanning physiological homeostasis and DSS-induced intestinal injury. Our study reveals that GLSP protects intestinal epithelia against chemical and age-related barrier injury by promoting the coordinated expansion of stem/progenitor compartments and restoring both the mucus and physical barriers. Gut microbiota involvement was confirmed via antibiotic-treated mice and fecal microbiota transplantation (FMT). FMT from GLSP-diet donors successfully recapitulated the protective effects in gut microbiota-depleted recipient mice. Multi-omics integration of 16S rRNA sequencing and metabolomic profiling identified the enrichment of SCFA-producing genera, and previously unrecognized microbial metabolites, such as indole-2-carboxylic acid, 3-Methyloxindole, and 4-hydroxyindole. These microbial metabolites are likely to play an essential role in reprogramming the intestinal metabolome, particularly tryptophan–indole metabolic pathways, to mediate intestinal homeostasis. These findings provide novel insights and potential directions for the development of new drugs targeting intestinal injury.