Conversion of cholesterol to bile acids in the liver is initiated by the rate-limiting enzyme cholesterol 7alpha-hydroxylase (CYP7A1) and excretion of bile acids from the liver is mediated by the bile salt export pump (BSEP). The expression of CYP7A1 and BSEP is coordinately regulated by a negative feedback and positive feed-forward mechanism, respectively, through bile acid-mediated activation of farsenoid X receptor (FXR). It is well established that hypolipidemic agent guggulsterone is an FXR antagonist and down-regulates FXR target genes. In this study, however, we have demonstrated that guggulsterone synergistically induced the expression of BSEP in cells treated with FXR agonist bile acids. A dissection study located in the BSEP promoter an activating protein (AP)-1 site supporting the action of guggulsterone. Deletion or mutation of the AP-1 element was diminished, whereas insertion of the AP-1 element into a heterologous promoter enhanced activation of the promoter by guggulsterone. Selective c-Jun N-terminal kinase and extracellular signal-regulated kinase inhibitors markedly decreased the transactivation, suggesting an involvement of AP-1 activation pathway in the up-regulation of BSEP by guggulsterone. Consistent with its FXR antagonism, guggulsterone antagonized bile acid-mediated transactivation of BSEP promoter when the AP-1 element was disrupted. In conclusion, guggulsterone regulates BSEP expression through composite mechanisms, and the transactivation through the AP-1 element is dominant over the FXR-mediated antagonism. The up-regulation of BSEP expression by guggulsterone without activating FXR pathway as an FXR agonist to suppress CYP7A1 expression represents a possible mechanism for guggulsterone-mediated hypolipidemic effect.