The purpose of this study was to elucidate the effects of dietary hemoglobin on serum and liver lipid contents in rats, and the ability of hemoglobin hydrolysates to disrupt lipid absorption. After rats had been fed on casein- or porcine hemoglobin-containing diets for 4 weeks, their serum and liver lipid contents and fecal cholesterol, bile acid, and nitrogen excretion were measured. To elucidate the mechanism of lipid absorption by dietary hemoglobin, we also examined lipase activity, micellar solubility of cholesterol, and bile acid binding activity in the presence of hemoglobin hydrolysates. Dietary hemoglobin decreased serum and liver triglyceride and cholesterol contents and increased fecal fatty acid, cholesterol, and bile acid excretion. In addition, hemoglobin hydrolysates inhibited lipase activity compared with casein hydrolysates in an in vitro study. These results suggested that the hypolipidemic effect of hemoglobin is mediated by increased fecal lipid excretion, and that decreased lipase activity by hemoglobin is at least partially responsible for this result. The observed effects were documented with an 8 g/kg hemoglobin diet, which is lower than in other studies; therefore. hemoglobin may be useful in the prevention of lifestyle-related diseases.