Cholelithiasis is a multifactorial process, and several mechanisms have been postulated. A decreased expression of the ileal apical sodium-dependent bile acid transporter (ASBT) and of the cytosolic ileal lipid binding protein (ILBP) was recently described in female non-obese patients. The role of the recently identified organic solute transporters alpha and beta (OSTalpha, OSTbeta) in gallstone pathogenesis remains unclear. Therefore, we performed analysis of OSTalpha-OSTbeta in gallstone patients according to body weight. Ileal mucosal biopsies were collected during routine colonoscopy from female gallstone carriers (n = 19) and controls (n = 34). OSTalpha-OSTbeta mRNA expression was measured using the LightCycler sequence detection system; protein was analyzed by immunohistochemistry and Western blot. The mRNA expression of OSTalpha-OSTbeta was significantly reduced (OSTalpha: 3.3-fold, P = 0.006; OSTbeta: 2.6-fold, P = 0.03) in normal-weight but not overweight gallstone carriers compared with controls. OSTalpha-OSTbeta protein levels also showed a reduction by 40-67%. The expression of OSTalpha-OSTbeta correlated positively with ASBT (r = 0.65, 0.58, respectively), ILBP (r = 0.77, 0.67), and the farnesoid X receptor (r = 0.58, 0.50). Fibroblast growth factor-19 showed a 2.8-fold reduction (P = 0.06), and liver receptor homolog-1 showed a 2-fold reduction (P = 0.04) in non-obese patients. In conclusion, an impaired function of all three ileal bile acid transporters may lead to low ileal bile acid reabsorption and an altered bile acid pool composition and therefore may contribute to the formation of gallstones in non-obese patients.