The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3.