The effects of sodium cholate on high-fat diet-induced hyperglycemia and obesity were investigated. Insulin resistance was estimated by measuring 2-deoxyglucose uptake in epitrochlearis muscles incubated in vitro. Addition of 0.5% cholate to high-safflower oil diet completely prevented high fat-induced hyperglycemia and obesity in C57BL/6J mice with a slight decrease of energy intake but with no inhibition of fat absorption. Furthermore, the addition of cholate decreased blood insulin levels and prevented high-fat diet-induced decrease of glucose uptake in epitrochlearis. However, there was no change in the unsaturation index of fatty acids in skeletal muscles and in GLUT-4 levels by cholate. In liver, cholate addition resulted in cholesterol accumulation and completely prevented high-fat diet-induced triglyceride accumulation. The changes of triglyceride level in the liver were paralleled to the changes of acyl-CoA synthetase (ACS) mRNA. ACS catalyzes the formation of acyl-CoA from fatty acid, and acyl-CoA is utilized for triglyceride formation in liver. ACS has a sterol-responsive element 1 in its promoter region. These data indicate that the favorable effects of cholate could be partly the result of downregulation of ACS mRNA.