Abstract: Objective: To explore the protective effects of 20-Hydroxyecdysone (20-HE) on high glucose induced HepG2 cells and its related molecular mechanism. Methods: In this study, high glucose (50 mmol/L glucose) was used to establish the oxidative damage model in HepG2 cells. The CCK-8 assay, caspase-3 assay, fluorescent probe method, and colorimetric method were used to assess the levels of cell viability, apoptosis, oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), respectively. The signaling pathways involved in the regulation of 20-HE were predicted using bioinformatics analysis. The phosphorylation level of Akt protein was detected by Western blot to evaluate the activation level of the PI3K/Akt signaling pathway. The involvement of the PI3K/Akt signaling pathway in the regulatory effects of 20-HE was verified using the inhibitor LY294002. Results: Treatment with 20-HE had no significant toxic effect on HepG2 cells at concentrations lower than 20 μmol/L. In the injured cells, 20-HE could significantly improve the viability (P<0.05), inhibit the apoptosis (P<0.05), down-regulate the level of ROS, improve the levels of SOD and CAT (P<0.05), and down-regulate the level of MDA (P<0.05). PI3K/Akt signaling pathway was the potential downstream mechanism of regulatory effects exerted by 20-HE. 20-HE could significantly up-regulate the level of PI3K/Akt signaling pathway in the injured cells (P<0.05). LY294002 could reverse the protective effects exerted by 20-HE on the injured cells. Conclusion: 20-HE exerted protective effects on high glucose induced oxidative damage in HepG2 cells by activating the PI3K/Akt signaling pathway.