Chiisanogenin (CHI), a bioactive compound derived from Eleutherococcus sessiliflorus (Rupr. & Maxim.), has gained attention for its potential therapeutic effects on glucose metabolism. This study aimed to elucidate the mechanisms underlying the blood glucose-lowering effects of CHI through both in vitro and in vivo investigations. In vitro experiments using L6 myotubes demonstrated that CHI enhanced glucose uptake in a dose-dependent manner. CHI promoted glucose transporter type 4 (GLUT4)-dependent glucose uptake through the insulin receptor substrate 1/phosphatidylinositol 3-kinase/protein kinase B (IRS-1/PI3K/Akt) signaling pathway, activating AS160 and facilitating the translocation of GLUT4 storage vesicles to the plasma membrane. Additionally, cell permeability assays using Caco-2 cells demonstrated that CHI possesses high cellular permeability. Mice treated with CHI before oral glucose administration showed a significant reduction in postprandial blood glucose levels compared to the control group. Overall, these results suggest that CHI activates insulin signaling independently of exogenous insulin and mimics insulin-like effects in vivo. Finally, molecular docking analysis was conducted to determine whether CHI interacts with the insulin receptor. The analysis predicted that CHI interacts with the insulin receptor with a binding energy of −6.96 kcal/mol. These findings suggest that CHI exerts a blood glucose-lowering effect, highlighting its potential as a functional material for the development of anti-diabetic functional food ingredient or dietary supplement after US FDA GRAS registration.