Sulfotransferase 1E1 (SULT1E1, also known as estrogen sulfotransferase) plays an important role in metabolism and detoxification of many endogenous and exogenous compounds (e.g., estrogens and flavonoids). Here we aimed to assess the effects of farnesoid X receptor (FXR) activation on SULT1E1 expression, and to determine the mechanism thereof. Treatment with specific FXR agonists (i.e., GW4064 and CDCA) significantly decreased both mRNA and protein levels of SULT1E1 in HepG2 cells. This was accompanied by a decrease in the enzymatic activity. The inhibitory effect was potentiated by FXR overexpression but attenuated by FXR knockdown, confirming FXR-dependent regulation of SULT1E1. Surprisingly, direct regulation of SULT1E1 by FXR was unlikely because FXR did not bind to SULT1E1 promoter or enhancer as revealed by chromatin immunoprecipitation (ChIP). Interestingly, SULT1E1 regulation was abolished when HNF4alpha (hepatocyte nuclear factor 4alpha, a known activator of SULT1E1) was silenced, supporting a critical role for HNF4alpha in FXR regulation of SULT1E1. Furthermore, a combination of ChIP, luciferase reporter and co-immunoprecipitation assays showed that FXR inhibited HNF4alpha transactivation of SULT1E1 by suppressed binding of the co-activator PGC1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha) to HNF4alpha. In conclusion, FXR transcriptionally regulates SULT1E1 through inhibition of PGC1alpha binding to HNF4alpha. Targeting the FXR-SULT1E1 axis may represent a promising approach for management of estrogen-related diseases.