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Saechalssal barley is Korea's representative naked waxy barley. This study investigated the anti-diabetic effect of the extract derived from saechalssal and its mechanism. The prethanol extract of saechalssal (SPE) showed greater α-glucosidase inhibitory activity in vitro and a more significant lowering of the postprandial blood glucose levels in normal mice compared to its water extract (SWE). When mice with type 2 diabetes (T2DM) induced by a high-fat diet and streptozotocin were fed SPE (200 mg/kg/day) for six weeks, the fasting blood glucose and serum free fatty acid levels were significantly lower than those of the control group. SPE significantly elevated the hepatic glycogen accumulation with increasing glycogen synthesis-related gene (GYS2 and UGP2) levels compared to the control group. SPE stimulated the expression of the hepatic glycolysis-related genes (GK, PFK1 , and PK) and suppressed the gluconeogenesis-related genes (G6Pase, FBP1 , and PEPCK). SPE up-regulated the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt), whereas it down-regulated the phosphorylation of glycogen synthase kinase 3 beta (GSK3β) compared to the control. The major flavonoids of SPE were naringin, prunin, and catechin, while its phenolic acids were ferulic acid and vanillic acid. These phytochemical compounds may contribute to the anti-hyperglycemic effects of SPE in diabetes. Overall, these results suggest that SPE has potential anti-diabetic activity through the regulating the PI3K/Akt/GSK3β pathway. [Display omitted] • Saechalssal prethanol extract inhibited α-glucosidase in vitro. • Saechalssal prethanol extract lowered fasting blood glucose in diabetic mice. • Saechalssal prethanol extract elevated hepatic glycogen accumulation in diabetic mice. • Saechalssal prethanol extract regulated glucose metabolism by PI3K/Akt/GSK3β pathway. [ABSTRACT FROM AUTHOR]