As a HDL receptor, SR-BI is a key regulator in enhancing reverse cholesterol transport in the liver, and hepatic overexpression of SR-BI can decrease plasma levels of HDL cholesterol, which may have anti-atherosclerosis effects. One of the mechanisms by which FXR is involved in regulating cholesterol and bile acid homeostasis is via transcriptional regulation of target gene expression. FXR has previously been shown to induce SR-BI expression. However, the underlying molecular mechanism by which FXR induces SR-BI expression is not fully defined. Therefore, the purpose of the current study is to determine the molecular mechanism by which FXR regulates SR-BI expression in human and mouse models. The present study showed that SR-BI mRNA was induced in a FXR-dependent manner in mouse livers, primary human hepatocytes and HepG2 cells. Our results suggest that FXR regulates Sr-bi gene expression by binding to multiple IR1s in the first intron of the Sr-bi gene. Moreover, the serum HDL level was increased in FXR-KO mice when fed either a control or HFD. Increased Sr-bi mRNA levels were shown to be FXR-dependent under HFD feeding by direct binding of FXR to the first intron of the Sr-bi gene, which indicates that FXR may enhance HDL uptake into the liver via inducing Sr-bi gene transcription. Activation of FXR has been demonstrated to regulate the expression of many hepatic genes involved in lipid homeostasis, including SR-BI. Consistent with our findings, Sr-bi mRNA levels have been shown to increase in livers of C57BL/6J mice but not in livers of FXR-KO mice upon LCA and CA feeding. However, a conflict result has been reported that Sr-bi expression was L-Ascorbyl 6-palmitate reduced following administration of CDCA or GW404764 both in vivo and in vitro. The authors further demonstrated that the decrease of Sr-bi was mediated by the FXR/RXR-SHP-liver receptor homologue 1 pathway. These conflicting results may be due to the different experimental models and/or ligands used. The underlying molecular mechanisms of FXR in regulating SR-BI and the role of SR-BI in FXR-mediated lipid homeostasis are still not clear. Recently, a vast database of nuclear receptor binding sites has been established with the development of genome-wide discovery of Terutroban transcription factor binding sites by ChIP-on-chip and ChIP-seq techniques. These data have shown that transcription factors tend to bind to multiple sites in the promoter and/or enhancer regions of target genes. Although our original ChIP-seq data did not show FXR binding to the promoter region of Sr-bi, we found three FXR binding sites within the first intron of Sr-bi gene. Moreover, even though luciferase activity did not correlate with the abundance of FXR binding, all three of these novel response elements, which include IR1s, were demonstrated functional.