-Klotho, a newly described membrane protein, regulates bile acid synthesis. Fibroblast growth factor 15 (FGF-15) and FGF receptor-4 (FGFR4) knockout mice share a similar phenotype with -Klotho deficient mice. FGF-15 secretion by the intestine regulates hepatic bile acid biosynthesis. The effects of -Klotho and FGF 15 on the ileal apical sodium bile transporter (ASBT) are unknown. -Klotho siRNA treatment of the mouse colon cancer cell line, CT-26, and the human intrahepatic biliary epithelial cells, HIBEC, resulted in up-regulation of endogenous ASBT expression that was associated with reduced expression of the farnesoid x-receptor (FXR) and the short heterodimer partner (SHP). Silencing -Klotho activated the ASBT promoter in CT-26, Mz-Cha-1 (human cholangiocarcinoma) and HIBEC cells. Site-directed mutagenesis of liver receptor homologue-1 (mouse) or retinoic acid receptor/retinoid X receptor (RAR/RXR) (human) cis-elements attenuated the basal activity of the ASBT promoter and abrogated its response to -Klotho silencing. siSHP, siFXR or dominant negative FXR treatment also eliminated the -Klotho response. FGF-15 secretion into cell culture media by CT-26 cells was diminished after siFGF-15 or si-Klotho treatment and enhanced by chenodeoxycholic acid. Exogenous FGF-19 repressed ASBT protein expression in mouse ileum, gallbladder and in HIBEC and repressed ASBT promoter activity in Caco-2, HIBEC and Mz-Cha-1 cells. Promoter repression was dependent upon the expression of FGFR4. These results indicate that both -Klotho and FGF15/19 repress ASBT in enterocytes and cholangiocytes. These novel signaling pathways need to be considered in analyzing bile acid homeostasis.