To define the biochemical and molecular basis of high-fat diet to reduce insulin secretion, the present study examined the effect of high-fat diet on the gene expression of pancreatic GLUT2, and glucokinase. Rats were isoenergetically meal-fed a high-fat or a high-carbohydrate diet for 10 weeks. Plasma glucose concentration was higher, whereas insulin concentration was lower in the rats fed a high-fat diet than in the rats fed a high-carbohydrate diet. The level of GLUT2 and glucokinase mRNA in pancreas was significantly decreased in the rats fed a high-fat diet compared with a high-carbohydrate diet. The rates of reduction in plasma insulin level, pancreatic gene expression of GLUT2 and glucokinase by high-fat feeding were about 50%. These results suggest that high-fat feeding impairs signal transduction mechanism in pancreatic β-cells to reduce insulin secretion in rats.