The somatostatin analog octreotide can lead to hyperbilirubinemia without evidence of liver injury. Here we investigate whether octreotide inhibits the main sinusoidal/canalicular bilirubin carriers and whether it is a transport substrate. Octreotide showed the most potent inhibitory effect toward OATP1B1-mediated transport and weaker inhibition for OATP1B3- and MRP2-mediated transport. Octreotide had no effect on OATP2B1-mediated transport. Octreotide inhibited [(3)H]estradiol-17-β-glucuronide (E17βG) influx mediated by OATP1B1, 1B3, and multidrug resistance-associated protein 2 (MRP2) in a concentration-dependent manner, and the IC50 values were computed to be 23 μM (95% confidence interval [CI] 18-29), 68 μM (95% CI 50-91), and 116.6 μM (95% CI 74.5-182.4), respectively. The interaction between octreotide and OATP1B1 was further studied. Inhibition of [(3)H]E17βG OATP1B1-mediated transport was purely competitive with no changes in maximum transport capacity (Vmax) and a twofold Km increase when the influx kinetics of [(3)H]E17βG were measured in the presence of octreotide (8.8 ± 3.1 versus 4.4 ± 1.2 μM, P = 0.03). The inhibition constant (Ki) of octreotide for the transport of [(3)H]E17βG was calculated at 33.5 ± 5.5 μM. Uptake of radiolabeled octreotide by OATP1B1-CHO cells was higher than in wild-type CHO cells and nonlabeled octreotide at the extracellular compartment was able to trans-stimulate the OATP1B1-mediated efflux of intracellular [(3)H]E17βG, suggesting that octreotide is a substrate of OATP1B1. In summary, this study shows interaction of octreotide on the human hepatocellular bilirubin transporters OATP1B1, OATP1B3, and MRP2, notably OATP1B1. These findings are in line with the clinical observation that a fraction of patients under treatment with octreotide exhibit hyperbilirubinemia.