Abstract: This study aimed to investigate the regulatory effects of stevia isochlorogenic acid (SICA) on lipid metabolism disorders and intestinal flora in high-fat diet-induced hyperlipidemic mice. Mice were divided into a blank control group, model group, simvastatin group, and low- and high-dose SICA groups. After 8 weeks of intervention, body weight was monitored regularly. Serum lipid levels, visceral fat changes, and the activity of key lipid-regulating enzymes in the liver were measured post-experiment. Pathological changes in the liver and adipose tissue were observed. Fecal samples were analyzed via 16S rRNA high-throughput sequencing to evaluate the effects of SICA on the composition, relative abundance, and diversity of gut microbiota. The results demonstrated that SICA significantly reduced weight gain in high-fat mice (P<0.01), decreased serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and leptin (P<0.01), and increased high-density lipoprotein cholesterol (HDL-C) and adiponectin levels (P<0.05). Furthermore, SICA inhibited the activity of hepatic fatty acid synthase (FAS) and β-hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase (P<0.01). Gut microbiota analysis revealed that SICA significantly elevated the Bacteroidetes/Firmicutes ratio and Shannon diversity index (P<0.01), while enriching functional genes associated with lipid metabolism. This study suggests that SICA effectively alleviates hyperlipidemia in high-fat diet-induced mice by reducing obesity, lipid accumulation, and hepatic lipid deposition. The underlying mechanism may involve the regulation of key enzymes in lipid metabolism pathways and modulation of gut microbiota composition, particularly by enhancing the Bacteroidetes/Firmicutes ratio and microbial diversity. These findings provide a theoretical foundation for the high-value utilization of Stevia byproducts and the development of natural lipid-lowering products.