Objective. To investigate the protective effect and mechanism of astragaloside IV (AS-IV) on damage in human glomerular endothelial cells (GEnCs) stimulated by high glucose and high insulin. Methods. The transwell method was used to detect the integrity of the cell barrier after AS-IV intervention in a high glucose and high insulin environment for 24 h; immunofluorescence and Western blot methods were used to detect the tight junction protein ZO-1 and claudin-5 expression; intracellular and extracellular 1β (IL-1β) and tumor necrosis factor α (TNFα) were determined by ELISA; expression and activation of AKT, p-AKT, GSK3α/β, and p-GSK3α/β were evaluated by Western blot. Results. The results showed that AS-IV had a significant protective effect on the cell barrier of GEnCs. High glucose or insulin inhibited cell viability in a concentration-dependent manner. High glucose or insulin significantly inhibited glucose uptake and promoted release of reactive oxygen species in GEnCs. Administration with AS-IV dramatically preserved viability of the cells; moreover, the expression of intracellular tight junction proteins was upregulated, inflammatory cytokines including IL-1β and TNFα were decreased, and the AKT-GSK3 pathway participated in modulation of AS-IV in GEnCs cells. Conclusion. We found in the present study that AS-IV can preserve filtration barrier integrity in glomerular endothelial cells under diabetic settings, its effects on increasing the cell energy metabolism and cell viability, inhibiting inflammation and oxidative stress damage, and enhancing tight junction between cells play a role in it; and the intracellular signaling pathway AKT-GSK modulated the above function. Our present finding supplied a new understanding towards development of DN and provided an alternative method on ameliorating DN.