Mice deficient in the organic solute transporter (Ost)-alpha subunit of the heteromeric organic solute and steroid transporter, Ostalpha-Ostbeta, were generated and were found to be viable and fertile but exhibited small intestinal hypertrophy and growth retardation. Bile acid pool size and serum levels were decreased by more than 60% in Ostalpha-/- mice, whereas fecal bile acid excretion was unchanged, suggesting a defect in intestinal bile acid absorption. In support of this hypothesis, when [3H]taurocholic acid or [3H]estrone 3-sulfate were administered into the ileal lumen, absorption was lower in Ostalpha-/- mice. Interestingly, serum cholesterol and triglyceride levels were also approximately 15% lower in Ostalpha-/- mice, an effect that may be related to the impaired intestinal bile acid absorption. After intraperitoneal administration of [3H]estrone 3-sulfate or [3H]dehydroepiandrosterone sulfate, Ostalpha-/- mice had higher levels of radioactivity in their liver and urinary bladder and less in the duodenum, indicating altered hepatic, renal, and intestinal disposition. Loss of Ostalpha was associated with compensatory changes in the expression of several genes involved in bile acid homeostasis, including an increase in the multidrug resistance-associated protein 3, (Mrp3)/Abcc3, an alternate basolateral bile acid export pump, and a decrease in cholesterol 7alpha-hydroxylase, Cyp7a1, the rate-limiting enzyme in bile acid synthesis. The latter finding may be explained by increased ileal expression of fibroblast growth factor 15 (Fgf15), a negative regulator of hepatic Cyp7a1 transcription. Overall, these findings provide direct support for the hypothesis that Ostalpha-Ostbeta is a major basolateral transporter of bile acids and conjugated steroids in the intestine, kidney, and liver.