Sulfation is an important pathway in the metabolism of thyroid hormone and estrogens. Sulfation of estrogens is reversible by estrogen sulfatase, but sulfation of thyroid hormone accelerates its degradation by the type 1 deiodinase in liver. Organic anion transporters (OATPs) are capable of transporting iodothyronine sulfates such as T4 sulfate (T4S), T3S, and rT3S or estrogen sulfates like estrone sulfate (E1S), but the major hepatic transporter for these conjugates has not been identified. A possible candidate is OATP1B1 because model substrates for this transporter include the bilirubin mimic bromosulfophthalein (BSP) and E1S, and it is highly and specifically expressed in liver. Therefore, OATP1B1-transfected COS1 cells were studied by analysis of BSP, E1S, and iodothyronine sulfate uptake and metabolism. Two Caucasian populations (155 blood donors and 1012 participants of the Rotterdam Scan Study) were genotyped for the OATP1B1-Val174Ala polymorphism and associated with bilirubin, E1S, and T4S levels. OATP1B1-transfected cells strongly induced uptake of BSP, E1S, T4S, T3S, and rT3S compared with mock-transfected cells. Metabolism of iodothyronine sulfates by cotransfected type 1 deiodinase was greatly augmented in the presence of OATP1B1. OATP1B1-Val174 showed a 40% higher induction of transport and metabolism of these substrates than OATP1B1-Ala174. Carriers of the OATP1B1-Ala174 allele had higher serum bilirubin, E1S, and T4S levels. In conclusion, OATP1B1 is an important factor in hepatic transport and metabolism of bilirubin, E1S, and iodothyronine sulfates. OATP1B1-Ala174 displays decreased transport activity and thereby gives rise to higher bilirubin, E1S, and T4S levels in carriers of this polymorphism.