Male Hartley guinea pigs were fed semipurified diets containing various levels of psyllium and cholesterol to determine mechanisms by which psyllium lowers plasma low density lipoprotein (LDL) concentrations. Four diets were tested: control diets with 12.5% (w/w) cellulose, and psyllium diets in which cellulose was partially replaced with 7.5% (w/w) psyllium. Two levels of dietary cholesterol were used, either low (LC, 0.04%, w/w) or high (HC, 0.25%, w/w). Plasma LDL was reduced by 30 and 54% with psyllium intake in the LC and HC groups, respectively (P < 0.001), while plasma very low density lipoprotein (VLDL) was lowered only in the HC group (P < 0.001). Psyllium intake modified LDL composition and size compared to LDL from control animals with a lower proportion of cholesteryl ester and higher proportion of triacylglycerol, lower molecular weight, smaller diameter, and higher peak density (P < 0.001). Plasma VLDL from animals fed the psyllium-HC diet compared to the control-HC contained lower relative proportions of free and esterified cholesterol and a higher proportion of triacylglycerol, compositional characteristics similar to VLDL from animals fed LC diets. Hepatic free and esterified cholesterol concentrations were significantly reduced by psyllium an average of 25 and 55%, respectively, while hepatic HMG-CoA reductase activity was increased in both psyllium groups compared to the respective controls (P < 0.001). In addition, psyllium intake reduced hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity in both the LC and HC groups (P < 0.001) and increased hepatic membrane apoB/E receptor number (Bmax) by 17 and 52% for animals fed LC and HC diets, respectively (P < 0.005). Significant psyllium-induced increases in cholesterol 7 alpha-hydroxylase of 4- and 1.6-fold were also observed in animals fed the LC and HC diets respectively (P < 0.001). These results indicate that psyllium generates a negative cholesterol balance across the liver which results in induction of cholesterol 7 alpha-hydroxylase and HMG-CoA reductase and suppression of ACAT activities, upregulation of apoB/E receptors, and secretion of smaller VLDL particles, metabolic alterations that contribute to a lowering of plasma LDL cholesterol levels.