Recently obeticholic acid (OCA) which is a farnesoid X receptor (FXR) agonist was approved by FDA to treat cholestatic liver diseases, which provided us a novel therapeutic strategy against cholestasis. Herein, we used a novel computational strategy with two-dimensional virtual screening for FXR agonists. For the first time, we found that auraptene (AUR), a natural product, can activate FXR to exert hepatoprotective effect against cholestatic liver injury in vivo and in vitro. Importantly, AUR was found to significantly decrease the mortality of cholestatic mice. Dynamic change analysis of bile acids and gene analysis revealed that AUR promoted bile acid efflux from liver into intestine via an induction in FXR-target genes Bsep and Mrp2 expression, and reduced hepatic uptake through an inhibition in Ntcp. Furthermore, AUR reduced bile acid synthesis through repressing FXR-target genes Cyp7a1 and Cyp8b1, and increased bile acid metabolism through an induction in Sult2a1. In addition, AUR promoted liver repair through an induction in liver regeneration-related gene, and suppressed liver inflammation through repressing inflammation-related gene NF-κB, TNF-α, IL-1β and IL-6. However, the changes in these genes and protein, as well as ameliorative liver histology induced by AUR were abrogated by FXR antagonist guggulsterone in vivo and FXR siRNA in vitro. These findings suggest that AUR may be an effective approach for the prevention against cholestatic liver diseases.