Chronic glucocorticoid overexposure predisposes to the development of atherosclerotic cardiovascular disease in humans. Cholestatic liver disease is associated with increased plasma glucocorticoid levels. Here, we determined – in a preclinical setting – whether the chronic presence of cholestatic liver disease also induces a concomitant negative impact on atherosclerosis susceptibility. Hereto, regular chow diet-fed atherosclerosis-susceptible hypercholesterolemic apolipoprotein E (APOE)-knockout mice were treated with the bile duct toxicant alpha-naphthylisothiocyanate (ANIT) for 8 weeks. ANIT exposure induced the development of fibrotic cholestatic liver disease as evident from collagen deposits and compensatory bile duct hyperproliferation within the liver and the rise in plasma levels of bilirubin (+60%; P < 0.01) and bile acids (10-fold higher; P < 0.01). Adrenal weights (+22%; P < 0.01) and plasma corticosterone levels (+72%; P < 0.01) were increased in ANIT-treated mice. In contrast, atherosclerosis susceptibility was not increased in response to ANIT feeding, despite the concomitant increase in plasma free cholesterol (+30%; P < 0.01) and cholesteryl ester (+42%; P < 0.001) levels. The ANIT-induced hypercorticosteronemia coincided with marked immunosuppression as judged from the 50% reduction (P < 0.001) in circulating lymphocyte numbers. However, hepatic glucocorticoid signaling was not enhanced after ANIT treatment. It thus appears that the immunosuppressive effect of glucocorticoids is uncoupled from their metabolic effect under cholestatic disease conditions. In conclusion, we have shown that cholestatic liver disease-associated endogenous glucocorticoid overexposure does not increase atherosclerosis susceptibility in APOE-knockout mice. Our studies provide novel preclinical evidence for the observations that the hypercholesterolemia seen in cholestatic human subjects does not translate into a higher risk for atherosclerotic cardiovascular disease.