The aim of this study was to explore in vitro glucose uptake activity in L6 cell lines and anti-diabetic activity of methanolic leaf extract of Acanthus ilicifolius (MLEA) in streptozotocin (STZ) induced type II diabetic rats. In vitro cytotoxicity and glucose uptake assay were carried out in L6 skeletal muscle cells using standard protocols. In vivo acute toxicity study was performed for 14 days. Type 2 diabetes was confirmed after 4 days of single intraperitoneal injection of STZ (40mg/kg bw) in albino Wistar rats. MLEA (100, 200 mg/kg bw) and metformin (500 mg/kg bw) were administered orally for 28 days. Body weight and glucose level from tail vein were monitored at regular interval. After 48 days, overnight fasted rats were sacrificed and blood was collected for further biochemical analysis. MLEA did not show any mortality up to 3000 mg/kg bw. Diabetic rats showed significant changes in fasting blood glucose, HbA1C, when compared with diabetic control group. There was significant elevation in body weight of diabetic treated rats. Thus, it was concluded that MLEA triggers glucose uptake in L6 cell lines and possesses antidiabetic activity in STZ induced diabetic rats.