Hepatocyte nuclear factor 4-alpha (HNF4α, NR2A1) is a nuclear receptor (NR) required for liver development and for controlling the expression of many hepatic-specific genes associated with important metabolic pathways. Many studies have also identified HNF4α as a direct transactivator of numerous xenobiotic-metabolizing cytochrome P450 (CYP) genes, suggesting that this factor is a global regulator which supports CYP transcription in the liver. Moreover, HNF4α expression displays a significant variability in human liver which may account for a proportion of the inter-individual variability in the expression of drug-metabolism genes and the clearance rate of a wide variety of prescribed drugs. In the last few years, a number of complex interactions and cross-talks between HNF4α and other transcription factors and coregulators have also surfaced, and the impact on CYP gene expression has been demonstrated. Thus, it is now clear that HNF4α modulates CYP expression in the liver by interacting with the xenosensor receptors (PXR and CAR), the glucocorticoid receptor (GR), the feeding-fasting cycle target PGC-1α, the sexual-dimorphism factor Stat5b, and other liver-enriched factors, such as C/EBPs. In addition to regulating drug elimination pathways, HNF4α also triggers pleiotropic effects on cholesterol and fatty acid metabolism, glucose homeostasis and inflammation. As a whole, current evidence indicates that HNF4α is a central regulator in the network of NRs that integrates drug-metabolism not only with the liver intermediate metabolism, but also with a number of patho-physiological conditions where the CYP expression is altered. The purpose of this review is to summarize and discuss these studies and their conclusions, with particular emphasis on the role of HNF4α in the regulation of drug-metabolizing CYP genes in the human liver.