Dietary soluble fiber (SF) consistently lowers plasma LDL cholesterol (LDL-C) concentrations, however, secondary mechanisms governing this reduction are not completely defined. Moreover, these mechanisms appear to differ with gender. Male, female and ovariectomized (to mimic menopause) guinea pigs were used to assess effects of gender, hormonal status and SF on activity and expression of hepatic cholesterol 7alpha-hydroxylase (CYP7). Diets were identical except for fiber source (control 10% cellulose, SF 5% psyllium/5% pectin). SF intake resulted in 44% lower plasma total cholesterol, 51% lower plasma LDL-C and 22% lower plasma triacylglycerol (TAG) concentrations. However, ovariectomized guinea pigs fed either the control or SF diets, had the highest plasma LDL-C and TAG levels (P<0.01). SF altered hepatic cholesterol metabolism by effectively reducing hepatic free cholesterol, TAG and microsomal free cholesterol, while activity of CYP7, the rate-limiting enzyme of cholesterol catabolism, was up-regulated. Hepatic CYP7 mRNA abundance paralleled the increase in enzyme activity. Ovariectomized guinea pigs had lowest activity and expression of hepatic CYP7 even after intervention with SF. These results suggest that induction of hepatic CYP7 activity may account, in large part, for the hypocholesterolemic effect of SF. Gender and hormonal status influence metabolic responses to dietary SF with estrogen deprivation leading to the most detrimental lipid profile.