Farnesoid X receptor (FXR) is a bile acid-sensing nuclear receptor that controls bile acid homeostasis. It has been suggested that down-regulation of FXR contributes to the pathogenesis of an inherited disorder of bile secretion caused by mutations in ATP8B1. We have investigated the relationship between ATP8B1 knockdown and FXR down-regulation in the human hepatoblastoma cell line HepG2. Transfection of HepG2 cells with ATP8B1 small interfering RNA (siRNA) duplexes led to a 60% reduction in the endogenous levels of ATP8B1 mRNA and protein, and a concomitant decrease in FXR mRNA and protein content, as well as in FXR phosphorylation. This decrease was accompanied by a marked reduction in mRNA levels of a subset of FXR targets, such as bile salt export pump (ABCB11), small heterodimer partner, and uridine 5'-diphosphate-glucuronosyltransferase. ATP8B1 inhibition specifically targeted FXR, since mRNA expression of other prominent nuclear receptors, such as PXR and CAR, or liver-enriched transcription factors, such as HNF-1a and HNF-4a, was not altered. The expression of other key genes involved in bile acid synthesis, detoxification and transport also remained unchanged upon ATP8B1 knockdown. Supporting the specificity of the effect, siRNA-mediated silencing of ABCB11, whose defect is associated with another inherited disorder of bile secretion, did not affect FXR expression. Treatment with the synthetic FXR agonist GW4064 was able to partially neutralize ATP8B1 siRNA-mediated FXR down-regulation, and fully counteract inhibition of FXR target genes. Collectively these findings indicate that ATP8B1 knockdown specifically down-regulates FXR and this action can be circumvented by treatment with FXR agonists.