To explore the possible mechanism of berberine (BBR) improving insulin resistance. We divided forty male Wistar rats into normall-diet control group (NC, n=10) and high-fat diet-caused obese group (HF, n=30). After the obese model rats established successfully (28 weeks), the NC group (n=10) and the obese group (HF1, n=10) were sacrificed. The rest of obese rats were divided randomly into two groups: untreated group (HF2, n=10) and 200 mg.kg-1. d-1 berberine- intervened group (BBR, n=10). Two groups were sacrificed after intervening eight weeks. We detected kinds of indicators of all rats. Our main results showed: 1. The level of endotoxin(ET), escherichia coli and enterococcus in HF1 group were significantly higher than those in NC group (P<0.05). 2. The mRNA and protein expression of TLR-4 and TNF-α of liver increased significantly and the mRNA expression of insulin receptor (IRC) and IRS-1 of liver decreased significantly in HF1 group compared with those in NC group (P<0.05). 3. The level of ET, FPG, FINS, HOMA-IR, TG and LDL-C decreased significantly in BBR group compared with those in HF2 group (P<0.05). Bifidobacterium and lactobacilli increased significantly, enterococcus and escherichia coli decreased significantly in BBR group compared with those in HF2 group (P<0.05).The mRNA and the protein expression of TLR-4 and TNF-α of the liver decreased significantly and the mRNA expression of IRC and IRS-1 of the liver increased significantly in BBR group compared with those in HF2 group (P<0.05). 4. HE staining showed the liver cells were mild fatty degeneration in BBR group, but the liver cells were obviously vacuolation and fatty degeneration in HF2 group. Our study suggests that BBR maybe improve the insulin resistance and the one of possible mechanism may due to that BBR could restore the balance of gut microbiota, lower the level of ET, down-regulate the mRNA and protein expression of the TLR-4 and TNF-α of liver.