The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostα-Ostβ exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostα-Ostβ have not been investigated. To determine the role of Ostα-Ostβ in intestinal bile acid absorption, the Ostα gene was disrupted by homologous recombination in mice. Ostα⁻/⁻ mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostα⁻/⁻ vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostα⁻/⁻Mrp3⁻/⁻ mice. The bile acid pool size was significantly reduced (>65%) in Ostα⁻/⁻ mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostα⁻/⁻ mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostα-Ostβ is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.