In humans, UDP-glucuronosyltransferase 1A9 is known to glucuronidate numerous lipophilic substances of pharmacological and toxicological importance. Although it has been established that individuals vary in their capacity to express this detoxification enzyme, little is known about the mechanisms that dictate the regulation of UGT1A9. In particular, it is not understood why, while the proximal regulatory regions of the UGT1A7-10 gene cluster are highly similar, UGT1A9 is the sole hepatic isoform of the four. Recent data have suggested that the human UGT1A9 promoter is controlled by hepatocyte nuclear factor 4alpha. In this work, we confirm that the human UGT1A9 promoter can indeed be upregulated by human hepatocyte nuclear factor 4alpha in vitro. Our results, however, show that the previously-reported hepatocyte nuclear factor 4alpha-binding site only plays a minor role in this response. Instead, upregulation was found to require a more proximal response element, which was not preserved in the UGT1A7, UGT1A8 or UGT1A10 promoters. Furthermore, hepatocyte nuclear factor 4alpha-mediated transcription from the human UGT1A9 promoter was discovered to be entirely dependent on hepatocyte nuclear factor 1. We have established that two hepatocyte nuclear factor 1-binding elements are involved in this phenomenon, the more distal of which is unique to the UGT1A9 promoter. Interestingly, this second site had no significant role in hepatocyte nuclear factor 1alpha-mediated induction of the UGT1A9 promoter in vitro, yet was critical for upregulation by human hepatocyte nuclear factor 4alpha. The discovery of two unique and cooperative liver-enriched transcription factor binding sites in the UGT1A9 promoter is a significant step towards understanding the unique hepatic expression of UGT1A9 amongst the UGT1A7-10 gene cluster.