Oxysterol 7α-hydroxylase catalyzes hydroxylation of oxysterols and neurosterols and plays a role in the alternative bile acid synthesis pathway. This gene is widely expressed in many organs and peripheral tissues and may protect tissues from the toxicity of oxysterols. Mutation in CYP7B1 caused neonatal cholestasis. To examine the regulatory mechanisms governing CYP7B1 expression, the 5′ flanking sequence of the CYP7B1 was analyzed and revealed a CpG island of about 1.2 kb. Transient transfection assays of deletion mutants of the CYP7B1 promoter-luciferase reporter gene in human liver-derived HepG2, fibroblast NT1088, and human embryonic kidney 293 cell lines revealed that the region from −291 to +189 was critical for gene transcription. Three GC box sequences located between −25 and +10 were essential for basal transcription because mutations of these sequences markedly reduced promoter activity. Sp1 and Sp3 bound to these sequences as demonstrated by DNase I footprinting assays and electrophoretic mobility shift assay. Thus, regulation of CYP7B1 transcription by Sp1 may play a pivotal role in regulating oxysterol levels, which regulate cholesterol metabolism.