We previously showed that plasma cholesterol levels decreased following ingestion of a short-chain fatty acid (SCFA) mixture composed of sodium salts of acetic, propionic, and butyric acids simulating cecal fermentation products of sugar-beet fiber (SBF). In the present study, we investigated whether hepatic and small intestinal cholesterol synthesis is involved in the cholesterol-lowering effects of SCFA and SBF. In vitro (expt. 1) and in vivo (expt. 2) cholesterol synthesis rates and the diurnal pattern of SCFA concentrations in portal plasma (expt. 3) were studied in three separate experiments in rats fed diets containing the SCFA mixture, SBF (100 g/kg diet), or the fiber-free control diet. Cholesterol synthesis was measured using 3H2O as a tracer. The in vitro rate of cholesterol synthesis, measured using liver slices, was greater in the SBF group, but not in the SCFA group, than in the fiber-free control group. In contrast, the hepatic cholesterol synthesis rate in vivo was lower in the SCFA group, but not in the SBF group, than in the control group. The mucosal cholesterol synthesis rate for the whole small intestine was <50% of the hepatic rate. The rate in the proximal region was slightly but significantly lower in the SCFA group, and was significantly higher in the SBF group than in the fiber-free group. The rate in the distal small intestines was also significantly greater in the SBF group than in the fiber-free group. Plasma total cholesterol concentrations were lower in the SCFA and SBF groups than in the fiber-free group in both experiments 2 and 3. Diurnal changes in portal SCFA and cholesterol levels were studied in the experiment 3. SCFA concentrations increased rapidly after the start of feeding the SCFA diet, and changes in plasma cholesterol were the reciprocal of those observed in SCFA. These results show that a decrease in hepatic cholesterol synthesis rate mainly contributes to the lowering of plasma cholesterol in rats fed the SCFA mixture diet. Changes in portal SCFA and cholesterol concentrations support this conclusion. In SBF-fed rats, SCFA produced by cecal fermentation are possibly involved in lowering plasma cholesterol levels by negating the counteractive induction of hepatic cholesterol synthesis caused by an increase in bile acid excretion.