Cholesterol (CH) homeostasis in the liver is regulated by enzymes of CH synthesis such as 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR) and catabolic enzymes such as cytochrome P450, family 7, subfamily a, polypeptide 1 (CYP7A1). Since a circadian clock controls the gene expression of these enzymes, these genes exhibit circadian rhythm in the liver. In this study, we examined the relationship between a diet containing CH and/or cholic acid (CA) and the circadian regulation of Hmgcr, low density lipoprotein receptor (Ldlr), and Cyp7a1 gene expression in the mouse liver. A four-week CA diet lowered and eventually abolished the circadian expression of these genes. Not only clock genes such as period homolog 2 (Drosophila) (Per2) and brain and muscle arnt like protein-1 (Bmal1), but also clock-controlled genes such as Hmgcr, Ldlr, and Cyp7a1 showed a reduced and arrhythmic expression pattern in the liver of Clock-mutant mice. The reduced gene expression of Cyp7a1 in mice fed a diet containing CA or CH+CA was remarkable in the liver of Clock-mutants compared to wild-type mice, and high liver CH accumulation was apparent in Clock-mutant mice. In contrast, a CH diet without CA only elevated Cyp7a1 expression in both wild-type and Clock-mutant mice. The present findings indicate that normal circadian clock function is important for the regulation of CH homeostasis in the mouse liver, especially in conjunction with a diet containing high CH and CA.