Abstract: Objective To explore the possible mechanism of berberine improve insulin resistance in skeletal muscle of obese rats. Methods Forty Wistar rats were divided into normal control group (NC group, n=10) and high fat food group (HF group, n=30). After the obese model rats established successfully, the rats of NC group (n=10) and HF group (n=10) were sacrificed. The level of fasting plasma endotoxin (ET) was detected. The expression of Toll-like receptor 4 (TLR4) mRNA in skeletal muscle were detected by real time quantitative PCR. The expression of TLR4, IκB kinase β (IKKβ), phospho-IKKβ^Ser181 (p-IKKβ^Ser181), nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phospho-IRS-1^Ser307 (p-IRS-1^Ser307) and the tyrosine phosphorylation of IR and IRS-1 (p-IR^Tyr and p-IRS-1^Tyr) protein in skeletal muscle were detected by Western blot. The twenty HF group rats were randomly divided into two group: the fat model control group (FC group, n=10) were fed with high fat diet and distilled water. The fat berberine group (FB group, n=10) were fed with high fat diet and berberine. All rats were sacrificed after effective interventions of eight weeks. The same indexes as the first part of experiment were detected. Results The results showed that the level of ET was increased. The TLR4/IKKβ/NF-κB signaling pathway is activated and the expression of TNF-α was increased of the skeletal muscle in obese rats. However, the insulin signaling pathways of the skeletal muscle in obese rats was inhibited. Berberine could reduce the level of ET of obese rats, down-regulate the TLR4/IKKβ/NF-κB inflammation signaling pathway and improve insulin resistance of skeletal muscle in obese rats. Conclusion Our study revealed that berberine could reduce the level of ET of obese rats, down-regulate the TLR4/IKKβ/NF-κB inflammation signaling pathway in skeletal muscle and berberine can improve insulin resistance of skeletal muscle through inhibiting the active of the TLR4/IKKβ/NF-κB signaling pathway.