D-chiro-inositol (DCI) is an active compound in tartary buckwheat [Fagopyrum tataricum (L.) Gaench] with an insulin-like bioactivity. The present study was performed to (i) prepare DCI-enriched tartary buckwheat bran extract (TBBE), (ii) evaluate its acute toxicity in mice, and (iii) examine its blood glucose lowering activity in diabetic mice. It was found that steaming buckwheat bran in an autoclave at 1.6 MPa and 120 degrees C for 60 min could significantly enrich the DCI level in TBBE from 0.03 to 0.22% and further to 22% after passage of the TBBE through activated carbon and ion exchange resins. An acute toxicity test demonstrated that the LD 50 of TBBE was >20 g/kg of body weight in mice, suggesting that TBBE was in general nontoxic and safe in mice. Male KK-A(y) mice (type 2 diabetic) and C57BL/6 mice (the control) were used to investigate the antidiabetic activity of TBBE. In KK-A(y) mice, the blood glucose, plasma C-peptide, glucagon, total cholesterol, triglyceride, and blood urea nitrogen (BUN) levels were significantly higher than those in the C57BL/6 mice. In addition, KK-A(y) mice showed an obvious decrease in insulin immunoreactivity in the pancreas. The present study clearly demonstrated that oral administration of DCI-enriched TBBE could lower plasma glucose, C-peptide, glucagon, triglyceride, and BUN, improve glucose tolerance, and enhance insulin immunoreactivity in KK-A(y) mice.