Acanthus ilicifolius L. leaf is extensively used in the Indian and Chinese medicine systems to treat diabetes mellitus. In this study, the antidiabetic effect of vitexin isolated from A.ilicifolius leaf extract and their effect on GLUT-4 translocation and PPAR-γ expression was evaluated high-fat dietstreptozotocinocin (HFD-STZ) induced rats. In vitro antidiabetic effect of vitexin was investigated through glucose uptake activity in L6 (rat skeletal muscle) cell lines. Vitexin (10 and 20 mg/kg BW was administered orally to HFD-STZ-induced diabetic rats for 48 days. The effect of vitexin on body weight, fasting blood glucose, serum insulin, total protein, urea, creatinine, and liver enzymes was examined. GLUT-4 translocation and PPAR-γ expression were studied in the skeletal muscle and adipocytes of experimental rats. The interaction of vitexin with GLUT-4 and PPAR-γ was validated by molecular docking analysis. Vitexin signi cantly lowered the blood glucose and also normalized other biochemical parameters. Further, the treatment with vitexin up-regulates the mRNA expression of GLUT-4 and PPAR-γ in diabetic rats. In silico analysis also supports the promising interactions between vitexin and target proteins. These results explained that vitexin up-regulates the mRNA expression of GLUT-4 and PPAR-γ and enhanced the translocation of GLUT-4 which maintains glucose homeostasis. Thus vitexin can be a gold promising drug for type II diabetes mellitus in the future.