To characterize the impact of gut microbiota on host bile acid metabolism, we investigated the metabolic profiles of oxysterols and bile acids (BAs) in a conventional rat model (SD) (n=5) and its pseudo germ-free (GF) equivalent (n=5). GF rats were developed by the oral administration of bacitracin, neomycin and streptomycin (200 mg/kg, each) twice a day for 6 days. Urinary levels of oxysterols and bile acid metabolites were quantified using gas chromatography-mass spectrometry (GC-MS). The activity levels of enzymes involved in the bile acid metabolic pathway were determined through urinary concentration ratio between product to precursor. Cholic acid (CA) and ${alpha}$-/${eta}$-muricholic acid (${alpha}-{eta}$-MCA) were significantly elevated at pseudo germ-free condition. An increase of hydroxylase (cholesterol 7${alpha}$-hydroxylase, oxysterol $7{alpha}$-hydroxylase and cytochrome P450 scc) and a significant decrease of $7{alpha}$-dehydroxylase were observed. The urinary concentration ratio of primary bile acids, a marker for hepatotoxicity, increased in pseudo germ-free conditions. Therefore, it was found that gut microbiota could play a significant role in the bile acids homeostasis and metabolism.