Sterol 27-hydroxylase (CYP27A1) catalyzes the hydroxylation of cholesterol to 27-hydroxycholesterol (27-OHC) and regulates cholesterol homeostasis. In () double knockout (KO) mice fed with Western diet (WD), the atherosclerotic phenotype found in KO mice was reversed. As protective mechanism, up-regulation of and was proposed. heterozygote/ KO (het) mice, with reduced expression and normal levels of and , developed more severe lesions than KO mice. To analyze the contribution of to the protection of atherosclerosis development, was induced by Rifampicin (RIF) in KO and het mice. Males were fed with WD and treated daily with RIF (10 mg/kg ip) or vehicle for 4 weeks. Atherosclerosis was quantified in the aortic valve. Plasma lipids and 27-hydroxycholesterol (27-OHC), expression of cytochromes P450 and genes involved in cholesterol transport and bile acids (BAs) signaling in liver and intestine, and intestinal cholesterol absorption were analyzed. RIF increased expression of hepatic but not intestinal 4-fold in both genotypes. In KO mice treated with RIF, we found a 2-fold decrease in plasma cholesterol, and a 2-fold increase in high-density lipoprotein/low-density lipoprotein ratio and CY27A1 activity. Intestinal cholesterol absorption remained unchanged and atherosclerotic lesions decreased approximately 3-fold. In het mice, RIF had no effect on plasma lipids composition, CYP27A1 activity, and atherosclerotic plaque development, despite a reduction in cholesterol absorption. In conclusion, the antiatherogenic effect of induction by RIF was also dependent on expression.Cyp27a1/ Apolipoprotein E