Eurotium cristatum-Fermented White Tea (FWT) significantly alters white tea (WT) composition, increasing caffeine while decreasing polyphenols and amino acids. FWT effectively ameliorated dextran sulfate sodium (DSS)-induced murine colitis symptoms (reducing weight loss, colon shortening). Mechanistically, FWT suppressed TLR4/Myd88/NF-κB signaling and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) while upregulating tight junction proteins (ZO-1, occludin, claudin-1), MUC2, and E-cadherin. Single-cell/spatial transcriptomics revealed that FWT treatments augment enterocyte, goblet cell, and stem cell populations, optimize goblet function, restructure stem cell differentiation, and induce epithelial REG3B (antimicrobial) and LYPD8 (motility inhibitor), plus immunomodulator GM42418 lncRNA across cell types, repairing the barrier. FWT intervention was also associated with an increase in beneficial bacteria (Akkermansia, Lactobacillus, Bifidobacterium), restoration of microbiota balance, and elevated levels of short-chain fatty acids (SCFAs) and was associated with alterations in caffeine-related metabolite profiles. Collectively, these multi-scale changes correlate with the alleviation of UC, suggesting an integrated mechanism involving mucosal barrier repair, immune–stromal modulation, microbiota–metabolism regulation, and cellular reprogramming.

Eurotium cristatum (EC), the dominant fungus in Fuzhuan brick tea, has significant applications in food fermentation and pharmaceutical industries, exhibiting probiotic properties, but further investigation of its intestinal benefits is required. This study characterized the EC-520 strain through whole genome sequencing and evaluated its effects on rat colons using histomorphology, 16S rRNA sequencing, and untargeted metabolomics. The genomic analysis revealed that EC-520 possessed a 28.37 Mb genome distantly related to Aspergillus flavus. The 16S results demonstrated that EC-520 significantly increased the abundance of Bacteroidota (p < 0.05) while decreasing the Proteobacteria and Firmicutes/Bacteroidota ratio (the F/B ratio); at the genus level, it elevated Muribaculaceae and Clostridia_UCG-014 while reducing harmful bacteria. The metabolomic results showed that EC-520 also significantly altered tryptamine, caproic acid, isocaproic acid, and erucic acid (p < 0.05). Additionally, the Spearman’s correlation analysis revealed that Muribaculaceae_unclassified and Clostridia_UCG-014_unclassified were significantly positively correlated with tryptamine, caproic acid, isocaproic acid, and erucic acid. Therefore, this study suggested that EC-520 enhanced the colon barrier and increased the abundance of Muribaculaceae_unclassified and Clostridia_UCG-014_unclassified, thus promoting the secretion of tryptamine and affecting the release of 5-hydroxytryptamine (5-HT). It also promoted the secretion of certain fatty acids, enhancing the balance of the colonic microbiota. This study provides a new view for a comprehensive understanding of EC’s regulatory role in the colon.