Glucose is important for energy; however, excessive daily intake of sugar may act as a toxin inducing the body to become overweight or obese. High blood glucose level reduces secretion of insulin, and glucose toxicity worsens insulin resistance. We investigated the metabolic fate of excess glucose by changing glucose levels in MRC-5 fibroblasts. Uptake of glucose into fibroblasts, the first stage of glucose metabolism, was measured. Treatment of fibroblasts under diabetic conditions led to rapid glucose incorporation. Glucose was absorbed into the cell almost constantly and reached excessive levels, and its metabolism was assessed by ¹⁴CO₂ output from [U-¹⁴C] D-glucose, the glucose metabolism end product. When fibroblasts were cultured in the presence of high glucose levels, CO₂ production decreased significantly in comparison with normal glucose conditions. Glucose metabolism in the diabetic setting was not accompanied by an increase in glucose uptake. Diabetic patients exercise tight glycemic control to avert disorders from such glucose toxicity. Pyruvate dehydrogenase (PDH) activity is reduced in diabetes; therefore, we investigated the influence of thiamine on PDH activity and intracellular glucose concentration in fibroblast cells exposed to diabetic conditions. Thiamine reversed high glucose-induced PDH inhibition and prevented glucose accumulation. These results, taken together with those of our previous report, suggest that thiamine partially plays a role in modifying the metabolic fate of glucose and reducing glucose toxicity.