Constitutive androstane receptor (CAR; NR1I3) controls the metabolism and elimination of endogenous and exogenous toxic compounds by up-regulating a battery of genes. In this work, we analyzed the expression of human CAR (hCAR) in normal liver during development and in hepatocellular carcinoma (HCC) and investigated the effect of hepatocyte nuclear factor 4α isoforms (HNF4α1 and HNF4α7) on the hCAR gene promoter. By performing functional analysis of hCAR 5′-deletions including mutants, chromatin immunoprecipitation in human hepatocytes, electromobility shift and cotransfection assays, we identified a functional and species-conserved HNF4α response element (DR1: ccAGGCCTtTGCCCTga) at nucleotide −144. Both HNF4α isoforms bind to this element with similar affinity. However, HNF4α1 strongly enhanced hCAR promoter activity whereas HNF4α7 was a poor activator and acted as a repressor of HNF4α1-mediated transactivation of the hCAR promoter. PGC1α stimulated both HNF4α1-mediated and HNF4α7-mediated hCAR transactivation to the same extent, whereas SRC1 exhibited a marked specificity for HNF4α1. Transduction of human hepatocytes by HNF4α7-expressing lentivirus confirmed this finding. In addition, we observed a positive correlation between CAR and HNF4α1 mRNA levels in human liver samples during development, and an inverse correlation between CAR and HNF4α7 mRNA levels in HCC. These observations suggest that HNF4α1 positively regulates hCAR expression in normal developing and adult livers, whereas HNF4α7 represses hCAR gene expression in HCC. (HEPATOLOGY 2007;45:1146–1153.)