Background: In addition to its LDL-lowering effect, bile salt (BS) sequestration with Colesevelam HCl has recently been shown to improve glycaemic control in patients with type 2 diabetes. Interference with BS signaling through the nuclear receptor Fxr during resin therapy has been postulated to affect hepatic lipogenesis and plasma triglyceride levels. Yet, whether BS sequestration actually reduces BS pool size and alters BS signaling functions in vivo is not known. Methods: Lean C57Bl/6J and diabetic db/db mice were fed standard chow without or with Colesevelam HCl (2% w/w) for two weeks. Kinetic parameters of BS metabolism were assessed by stable isotope dilution using [2H4]cholate. Lipogenesis was determined from incorporation of [1-13C]-acetate (2% w/v in water) followed by MIDA analyses. Gene expression patterns were assessed by Q-PCR Results: Enhanced cholate synthesis (+375% and +172%, lean and db/db mice, respectively) fully compensated for its enhanced fractional turnover (3-fold in both strains) upon Colesevalam HCl-treatment in both groups: cholate pool size remained unaffected. Consequently, the transhepatic BS flux remained virtually unchanged and no indications for altered Fxr-activity were observed in either group. Yet, both lean (+50%) and db/db (+23%) mice showed significantly increased hepatic triglyceride contents upon resin feeding with a consistently increased hepatic expression of lipogenic genes (Srebp1c, Acc1, Fas, Scd1) and a clear induction of de novo lipogenesis. Complete absence of intestinal expression of Fgf15 in Colesevelam HCl-treated mice likely contributes to massively induced hepatic BS synthesis and might be involved in adaptations of hepatic lipid metabolism. Conclusion: Since BS pool size and transhepatic flux remained unaffected, induction of the hepatic lipogenic pathway of Colesevelam HCl-treated mice likely involves hepatic Fxr-independent mechanisms.