To establish an insulin-resistant Hep G2 cell model and observe the protective effect and mechanism of Isaria feline (IF) on oxidative stress of insulin-resistant Hep G2 cells. Methods The proliferation of Hep G2 cells was determined by MTT assay. Hep G2 cells were induced by high-glucose DMEM medium containing 10-8 mol·L-1 insulin for 48 h to establish insulin-resistant Hep G2 cell model. Glucose residue, superoxide dismutase (SOD), malondialdehyde (MDA), IRS-1, PI3K and Akt were determined by kit. Results The concentration of 5μg·mL-1 and 10μg·mL-1 of IF did not significantly promote the cell growth, and the insulin concentration was 10-8 mol·L and cultured for 48h to establish the insulin resistance model. Compared with normal group, the glucose consumption of Hep G2 cells in model group decreased significantly (P＜0.01), SOD content decreased significantly (P＜0.01), MDA content increased significantly (P＜0.01), the content of IRS-1, PI3K and Akt decreased significantly (P＜0.01,P＜0.01,P＜0.05). After treatment, glucose consumption was increased significantly (P＜0.01), SOD content increased significantly (P＜0.01), MDA content decreased significantly (P＜0.01), the content of IRS-1、PI3K and Akt were increased significantly (P＜0.01,P＜0.05,P＜0.01).Conclusion IF can improve glucose metabolism and oxidative stress levels in insulin-resistant Hep G2 cell model, and alleviates insulin resistance by regulating IRS-1/PI3K/Akt signaling pathway