The present study was carried out to investigate the antiobesity and hypocholesterolemic effects of Bifidobacteria animalis DY-64 (B. animalis DY-64), a lactic acid bacterium isolated from the human intestine, in rats fed a high-fat/high-cholesterol diet for 4 weeks. Forty male Sprague-Dawley rats were divided into four groups and fed either a normal (N) or highfat/ high-cholesterol (HFC) diet without or with oral administration of B. animalis DY-64 (N-BA, HFC-BA). The gain in body weight, and liver and adipose tissue weights of the HFC group were heavier than that of the HFC-BA group. Serum total cholesterol (TC), LDL-cholesterol, and leptin levels of the HFC group, which were significantly elevated compared to those of the N group, dropped by 19, 18, 21, and 13% in the HFC-BA group, respectively, whereas the serum HDL-cholesterol level markedly increased. However, serum TG, LDL-cholesterol, HDL-cholesterol, and leptin levels were not significantly different among the N groups (N, N-BA) with or without B. animalis DY-64 administration. TC and TG levels of the liver as well as the TG level of the adipose tissue were significantly reduced in the HFC-BA group. In addition, HR-LPL activity in adipose tissue was also lower in the HFC-BA group than in the HFC group. These results suggest that B. animalis DY-64 isolated from the human intestine exerts hypocholesterolemic effects by reducing serum and liver cholesterol levels and plays a role in the prevention of obesity induced by HFC diet.