Recent data concerning the effect of soybean 7S globulin subunits on the upregulation of LDL receptors in Hep G2 cells identified the α′ subunit as the candidate responsible for this biological effect. In vivo evaluation of this subunit on cholesterol homeostasis was hampered by the lack of suitable amounts of α′ chain. A novel separation procedure allowed us to investigate the effects of α′ subunit administration on plasma cholesterol and triglyceride levels, as well as on the activity of liver β-VLDL receptors of rats fed a hypercholesterolemic (HC) diet. Rats were divided into 9 groups fed the following diets for 28 d: standard diet; HC diet; HC diets + 5, 10, and 20 mg/(kg body weight · d) of α′ subunit; HC diets + 50, 100, and 200 mg/(kg body weight · d) of soybean 7S globulin; HC diet + 200 mg/(kg body weight · d) clofibrate. The highest dose of the α′ subunit decreased plasma cholesterol and triglycerides 36 and 34%, respectively, in rats fed the HC diet; 10-fold amounts clofibrate reduced plasma cholesterol and triglycerides 38 and 41%. The activity of liver β-VLDL receptors of rats fed the HC diet with the highest dose of the α′ subunit had a 96% increase in binding compared with the HC diet group, thus restoring the receptor activity to that of rats fed the standard diet. These results represent the first in vivo evidence of both the plasma lipid-lowering properties and the upregulation of liver β-VLDL receptors induced by the soybean α′ subunit. J. Nutr. 134: 1334-1339, 2004.