ABCG2, also known as breast cancer resistance protein or BCRP, is an efflux transporter that is involved in the excretion of various compounds in multiple tissues and in the multidrug resistance in tumor cells. Its substrates include endobiotics such as estrone sulfate and urate, anticancer agents such as mitoxantrone, topotecan and methotrexate, and metabolites of environmental chemicals such as benzo[a]pyrene-3-sulfate and benzo[a]pyrene-3-glucuronide. Recent studies have reported that ABCG2 mRNA levels in cultured human hepatocytes were increased after treatment with phenobarbital and that treatment of epileptic patients with carbamazepine caused the increase in hepatic ABCG2 mRNA levels. These results suggest a role of constitutive androstane receptor (CAR) in the transcriptional activation of human ABCG2 (hABCG2). The precise mechanism, however, remains unknown. In the present study, hABCG2 mRNA levels in human hepatocytes were found to increase after infection with adenovirus expressing human CAR (hCAR) and treatment with its activator. Reporter assays using reporter plasmid containing various lengths of hABCG2 5’-flanking regions and hCAR expression plasmid suggested that an hCAR-responsive element is located between -8000 and -7485. With electrophoretic mobility shift assays and chromatin immunoprecipitation assays, a DR5 motif (direct repeat separated by 5 nucleotides), located at -7988 to -7972 in a 3’ to 5’ orientation, was identified as a binding motif of hCAR/human retinoid X receptor a heterodimer. The motif was termed G2-DR5. The introduction of mutations into G2-DR5 of the hABCG2 reporter genes resulted in the complete loss of the hCAR-mediated increase in reporter activities. In addition, hCAR transactivated the reporter construct containing three copies of wild-type G2-DR5, but not mutated G2-DR5, under thymidine kinase promoter. These results suggest a critical role of G2-DR5 in the hCAR-mediated transactivation of hABCG2. Interestingly, the extent of hCAR-mediated transactivation was higher with the construct containing three copies of G2-DR5 in a natural orientation (3’ to 5’) was higher than that containing complement G2-DR5 motifs. Finally, we found that human pregnane X receptor, which belongs to the same NR1I subfamily of the nuclear receptor superfamily as CAR, did not activate any reporter gene containing G2-DR5 in reporter assays. Taken together, our results suggest that in human hepatocytes hCAR transactivates hABCG2 through the DR5 motif located in the distal promoter of hABCG2 and that the motif is specific to CAR but not pregnane X receptor.