The present study investigated the anti-diabetic effects of new herbal formula (NHF) consist of Polygonati Rhizoma, Rehmanniae Radix, Salviae miltiorrhizae Radix, Puerariae Radix, Schizandrae Fructus, Glycyrrhizae Radix in neonatal streptozotocin (nSTZ)-induced non-insulin-dependent diabetes mellitus (NIDDM) rats. Changes of food and water intakes, body weight, blood glucose, plasma insulin and immunohistochemical evaluation of insulin on pancreas, and mRNA expression of glucose transporter subtype-4 (GLUT-4) in skeletal muscle and hepatic phosphoenolpyruvate carboxykinase (PEPCK) by administration of NHF (300 mg/kg) were investigated. The nSTZ diabetic rats showed hyperglycemia, increases in food and water intake, loss of body weight gain and decrease of the number of insulin-positive cells and the size of beta-cells in pancreas and mRNA of GLUT-4 in soleus muscle and increase of hepatic PEPCK mRNA expression. Administration of NHF significantly decreased the blood glucose level, food and water intake and considerably increased the body weight of nSTZ diabetic rats. Also, NHF treatment significantly increased plasma insulin level and the number and size of insulin-immunoreactive cells in the pancreas of nSTZ diabetic rats. In addition, NHF treatment resulted in increased expression of the GLUT-4 mRNA in soleus muscle and in reduced expression of PEPCK mRNA in liver. These results provide possible mechanisms for the anti-diabetic effects of NHF, via a decrease of blood glucose level, an increase of insulin sensitivity, an increase of GLUT-4 gene expression and an attenuation of hepatic PEPCK gene expression. In conclusion, NHF may be useful for improving hyperglycemia and reducing the risk of diabetic complications.