The chipmunk hibernation-specific HP-27 gene is expressed specifically in the liver and has a CpG-poor promoter. To reveal how the liver-specific transcription of the HP-27 gene is regulated, we performed yeast one-hybrid screening of a chipmunk liver cDNA library. A 5'-flanking sequence of the HP-27 gene, extending from -170 to -140 and containing an E-box (5'-CACGTG-3'), is essential for the liver-specific transcription of HP-27. We used this sequence as bait and found that a ubiquitously expressed transcription factor, USF (upstream stimulatory factor), bound to the E-box. In COS-7 cells, USF activated transcription from the HP-27 gene promoter. We then used bisulphite genomic sequencing to analyse the methylation status of the four CpG dinucleotides that lie in the 5'-flanking sequence of the HP-27 gene up to -450, to investigate how the ubiquitously expressed USF activates transcription of the HP-27 gene only in the liver, while its transcription is repressed elsewhere. The only difference in methylation in the tissues tested was in the CpG dinucleotide in the USF-binding site, which was hypomethylated in the liver, but highly methylated in the kidney and heart. The specific methylation of the CpG dinucleotide at the USF-binding site impeded both the binding of USF and its transcriptional activation of the HP-27 gene. Chromatin immunoprecipitation using anti-USF antibodies revealed that USF bound to the HP-27 gene promoter in the liver, but not in the kidney or heart. Thus CpG methylation at the USF-binding site functions in establishing and maintaining tissue-specific transcription from the CpG-poor HP-27 gene promoter.