AIMS: Here, regulatory effects of Xiaoyaosan polysaccharide on entire intestinal flora and butyrate-producing bacteria were investigated to reveal their pharmacological mechanism serving as bacterial-derived carbon sources for regulating intestinal microecology during the treatment of chronic unpredictable mild stress (CUMS)-induced depression in rats. METHODS AND RESULTS: The effects were measured by analyzing depression-like behavior, intestinal flora, butyrate-producing bacteria diversity, and fecal butyrate content. After intervention, CUMS rats exhibited alleviated depression and increased body weight, sugar water consumption rate, and performance index in the open-field test (OFT). The abundance of dominant phyla, such as Firmicutes and Bacteroidetes, and dominant genera, such as Lactobacillus and Muribaculaceae, was regulated to restore the diversity and abundance of the entire intestinal flora to a healthy level. The polysaccharide enriched the diversity of butyrate-producing bacteria, increased the abundance of the butyrate-producing bacteria Roseburia sp. and Eubacterium sp., reduced the abundance of Clostridium sp., increased the distribution of Anaerostipes sp., Mediterraneibacter sp., and Flavonifractor sp., and subsequently increased the content of butyrate in the intestine. CONCLUSIONS: These findings suggest that the Xiaoyaosan polysaccharide alleviates unpredictable mild stress-induced depression-like chronic behavior in rats by regulating the composition and abundance of the entire intestinal flora, restoring the diversity of butyrate-producing bacteria, and increasing the butyrate levels.