To elucidate whether a low methionine (Met)/glycine (Gly) ratio is responsible for the hypocholesterolemic effect of rice protein (RP), the effects of adding Met to RP, increasing its Met/Gly ratio, on hepatic cholesterol output was investigated in rats fed cholesterol-free diets. The hepatic secretion of cholesterol into bile or circulation was measured from isolated perfused livers of 7-week-old male Wistar rats fed RP (cultivar Koshihikari) with a lower Met/Gly ratio and methionine-supplemented RP with a higher Met/Gly ratio (RP-M) matched with casein (CAS). RP-M produced a significant hypocholesterolemic effect, whereas the effect of RP on plasma cholesterol level was comparable to that of CAS. Hepatic accumulation of total lipids, cholesterol, and phospholipids was higher in RP-M rats than in RP rats, again following a pattern similar to that in CAS rats. The hepatic total and very-low-density lipoprotein (VLDL)-cholesterol secretions into circulation were effectively decreased by RP, but not by RP-M. Bile flow and biliary outputs of bile acids, cholesterol, and phospholipids were significantly stimulated by RP-M, causing an increase in fecal sterol excretion compared with CAS and RP. Neither biliary output nor fecal excretion of bile acids was affected by RP compared with CAS. The present results demonstrate that the plasma cholesterol-lowering effect of RP cannot be merely ascribed to the low Met/Gly ratio of RP in growing rats fed cholesterol-free diets. Results suggest that the hypocholesterolemic response induced by RP-M with a higher Met/Gly ratio primarily contributes to the stimulation of hepatic cholesterol for elimination via biliary secretion rather than the inhibition of hepatic cholesterol release via VLDL into circulation.