The cholesterol-lowering efficacy and possible mechanism of action of polymethoxylated flavones (PMFs) derived from Ortanique peel (PMFort) were evaluated in diet-induced hypercholesterolemic rats. Hypercholesterolemic and normal rats were fed diets containing PMFort and niacin for 49 days, after which several biochemical parameters were determined in the serum and organ samples. Diets containing 1.5% PMFort significantly reduced serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C) and triglyceride (TG) levels by 45%, 69%, 30% and 24%, respectively, and significantly increased serum high-density lipoprotein cholesterol (HDL-C) levels by 45% compared to the untreated hypercholesterolemic rats. Fecal cholesterol was also significantly increased (106%) by PMFort supplementation. Hepatic cholesterol was significantly reduced as well as the hepatic activity of HMG-CoA reductase in the PMFort supplemented group compared to the untreated hypercholesterolemic groups. The findings suggest that PMFort may be a potent hypocholesterolemic supplement that acts by reducing cholesterol synthesis as well as by increasing fecal cholesterol output.