Caveolin-1 (CAV1) is the structural protein of the filamentous coat that decorates the cytoplasmic surface of each caveola. Cell culture studies have implicated CAV1 in playing an important role in intracellular cholesterol trafficking. In addition, it has been reported that CAV1 forms a detergent-resistant protein complex with Annexin-2 in enterocytes that can be disrupted by the cholesterol absorption inhibitor ezetimibe, suggesting a possible role for CAV1 in cholesterol absorption. In this report, we have evaluated cholesterol homeostasis in Cav1 knock-out mice. Deletion of CAV1 does not result in either a compensatory increase of CAV2 or CAV3 in intestine. In addition, Cav1 knock-out mice display normal mRNA and protein levels of Annexin-2 or the putative cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) in proximal intestinal mucosa. Fractional cholesterol absorption and fecal neutral sterol excretion are statistically similar in Cav1 knock-out mice and their wild-type littermates. Moreover, oral administration of ezetimibe is equally effective in decreasing cholesterol absorption in Cav1 null mice and wild-type controls. The mRNA expression levels of genes sensitive to intracellular cholesterol concentration (ATP-binding cassette transporters ABCA1 and ABCG5, hydroxymethylglutaryl-CoA synthase and the LDL receptor) are similarly altered in the proximal intestinal mucosa of Cav1 null and wild-type mice following ezetimibe treatment. These results demonstrate that CAV1 is not required for cholesterol absorption or ezetimibe sensitivity in the mouse.