The mechanisms responsible for bile acid regulation of mouse intestinal organic solute transporter α-β (Ostα-Ostβ) expression were investigated. Expression of Ostα-Ostβ mRNA was increased in cecum and proximal colon of cholic acid-fed mice and in chenodeoxycholate-treated mouse CT26 colon adenocarcinoma cells. Sequence analysis revealed potential cis-acting elements for farnesoid X receptor (FXR) and liver receptor homolog-1 (LRH-1) in the mouse Ostα and Ostβ promoters and reporter constructs containing Ostα and Ostβ 5′-flanking sequences were positively regulated by bile acids. Expression of a dominant-negative FXR, reduction of FXR with interfering small RNA (siRNA), or mutation of the potential FXR elements decreased Ostα and Ostβ promoter activity and abolished the induction by chenodeoxycolic acid. Negative regulation of the Ostα and Ostβ promoters by bile acids was mediated through LRH-1 elements. Ostα and Ostβ promoter activities were increased by coexpression of LRH-1 and decreased by coexpression of SHP. Mutation of the potential LRH-1 elements and siRNA-mediated reduction of LRH-1 expression decreased basal promoter activity. As predicted from the promoter analyses, ileal Ostα and Ostβ mRNA expressions were increased in wild-type mice administered the FXR agonist GW4064 and decreased in FXR-null mice. Immunoblotting analysis revealed that Ostα and Ostβ intestinal protein expressions correlated with mRNA expression. The mouse Ostα and Ostβ promoters are unusual in that they contain functional FXR and LRH elements, which mediate, respectively, positive and negative feedback regulation by bile acids. Although the positive regulatory pathway appears to be dominant, this arrangement provides a mechanism to finely titrate Ostα-Ostβ expression to the bile acid flux.