Animal studies suggest that propionate, derived from colonic carbohydrate fermentation, may be gluconeogenic and inhibit cholesterol synthesis in the liver. We therefore studied, in six healthy subjects, the effect of rectally infused solutions containing acetate alone (180 mmol), propionate alone (180 mmol), or a mixture of acetate (180 mmol) and propionate (60 mmol). Relative to the control infusion of normal saline, acetate increased serum cholesterol, glucagon, and acetate concentrations and reduced free fatty acids (FFAs) within 30 min. Propionate alone increased serum propionate, glucose, and glucagon with no effects on cholesterol and a delayed fall in FFAs. The addition of propionate to acetate resulted in no significant rise in serum cholesterol. These results are consistent with the hypothesis that colonic propionate is a gluconeogenic substrate in humans and inhibits the utilization of acetate for cholesterol synthesis.