Formononetin (FMNT), an isoflavone commonly found in medicinal and food-related plants, demonstrates promising therapeutic potential. In this study, FMNT treatment significantly improved depressive-like behaviors in rats, alleviated colonic tissue damage, and suppressed the release of peripheral lipopolysaccharide (LPS) and inflammatory factors. It also enhanced intestinal barrier function by increasing the expression of tight junction proteins (claudin-1, claudin-6, and occludin). Microbiota analysis revealed that FMNT mitigated LPS-induced dysbiosis, reducing Proteobacteria levels and restoring the Firmicutes/Bacteroidetes (F/B) ratio. At the genus level, FMNT increased the abundance of S24-7 and Lactobacillus while inhibiting Dorea. Moreover, FMNT modulated microbial metabolism by influencing pathways associated with lysine degradation, arginine biosynthesis, and steroid hormone biosynthesis, along with elevating short-chain fatty acids (SCFAs). Collectively, this study underscores FMNT’s vital role in ameliorating depressive-like symptoms triggered by neuroinflammation, primarily through the maintenance of intestinal homeostasis and the rebalancing of the gut microbiota.