Recurrent Clostridioides difficile infection (rCDI) is a global health threat and receives increased attention. Berberine (BBR), a natural pentacyclic isoquinoline alkaloid, has been used as a cost-effective treatment for intestinal infections in Asia for many years. However, its effect on rCDI is not clear yet. The efficacy and the underlying mechanisms of BBR were evaluated in vancomycin-dependent rCDI mouse model and intestinal organoids model. Our findings revealed that BBR treatment alleviated the severity and increased survival rate in rCDI mice. Mechanistically, BBR alleviated intestinal epithelial damage with higher Occludin expression, suppressed some inflammatory pathways and reduced the level of inflammatory factors in both cecum and serum. Moreover, the 16s rRNA sequencing analysis indicated that BBR reshaped the gut microbiota by increasing the abundance of Firmicutes and reducing the abundance of Proteobacteria. At genus level, BBR treatment increased levels of Blautia and Bilophila, and reduced levels of Proteus. In addition, acetic acid, one of the short-chain fatty acids (SCFAs), was also increased after BBR treatment in rCDI mice. Collectively, BBR exerted a protective effect in rCDI via multiple underlying mechanisms, which deserve further investigation as a potential drug candidate for alleviating rCDI.