Secretion of cholesterol into bile is important for the elimination of cholesterol from the body. Thyroid hormone (TH) increases biliary cholesterol secretion and hepatic gene expression of adenosine triphosphate (ATP)‐binding cassette, subfamily G (WHITE), member 5 (ABCG5) and ATP‐binding cassette, subfamily G (WHITE), member 8 (ABCG8), two half‐transporters that act as a heterodimeric complex promoting sterol secretion. In addition, nuclear liver x receptor‐alpha (LXRa), also regulated by TH, induces gene expression of ABCG5/G8. We here investigated if the TH‐induced stimulation of biliary cholesterol secretion is mediated by the ABCG5/G8 complex in vivo, and if so, whether LXRa is involved. Mice homozygous for disruption of Abcg5 (Abcg5^−/−) or Lxra (Lxra^−/−) and their wild‐type counterparts were treated with triiodothyronine (T3) for 14 days and compared to untreated mice of corresponding genetic backgrounds. Bile was collected by gallbladder cannulation, and liver samples were analyzed for gene expression levels. Basal biliary cholesterol secretion in Abcg5^−/− mice was 72% lower than in Abcg5^+/+ mice. T3 treatment increased cholesterol secretion 3.1‐fold in Abcg5^+/+ mice, whereas this response was severely blunted in Abcg5^−/− mice. In contrast, biliary cholesterol secretion in T3‐treated Lxra^+/+ and Lxra^−/− mice was increased 3.5‐ and 2.6‐fold, respectively, and did not differ significantly. Conclusions: TH‐induced secretion of cholesterol into bile is largely dependent on an intact ABCG5/G8 transporter complex, whereas LXRa is not critical for this effect. (HEPATOLOGY 2012;56:1828–1837)