Gut microbiota dysbiosis and gut barrier disruption are key events associated with high-fat diet (HFD)-induced systemic metabolic disorders. Gymnemic acid (GA) has been reported to have an important role in alleviating HFD-induced disorders of glycolipid metabolism, but its regulatory role in HFD-induced disorders of the gut microbiota and gut barrier function has not been elucidated. Here we showed that GA intervention in HFD-induced hamsters increased the relative abundance of short-chain fatty acid (SCFA)-producing microbes including Lactobacillus (P<.05) and Lachnoclostridium (P<.01) in the gut, and reduced the relative abundance of lipopolysaccharide (LPS)-producing microbes including Enterococcus (P<.05) and Bacteroides (P<.05), subsequently improving HFD-induced intestinal barrier dysfunction and systemic inflammation. Specifically, GA intervention reduced mRNA expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α (P<.01), increased mRNA expression of antioxidant-related genes, including Nfe2l2, Ho-1, and Nqo1 (P<.01), and increased mRNA expression of intestinal tight junction proteins, including Occludin and Claudin-1 (P<.01), thereby improving gut barrier function of HFD hamsters. This ameliorative effect of GA on the gut of HFD hamsters may further promote lipid metabolic balance in liver and adipose tissue by regulating the Toll-like receptor 4 (TLR4)-nuclear factor-κB (NF-κB) signaling pathway. Taken together, these results systematically revealed the important role of GA in regulating HFD-induced gut microbiota disturbance and gut barrier function impairment, providing a potential clinical theoretical basis for targeted treatment of HFD-induced microbiota dysbiosis.