25-Hydroxycholesterol (25HC) is the product of the cholesterol 25-hydroxylase (CH25H) gene, and is one of the most potent inhibitors of cholesterol synthesis, through the suppression of SREBP activation. 25HC has also been implicated in neurodegenerative disease and shown to be toxic to primary cultured neuronal cells. Very little is known about the transcriptional regulation of the CH25H gene. Using in silico analysis of the mouse and human promoters, potential binding sites for several transcription factors were identified including p53, CREB and Sp1. The functionality of the putative promoter elements of the mouse promoter was investigated using an in vitro reporter assay with deletion constructs. The results suggest the proximal CREB binding site may be involved in driving expression from the CH25H promoter. While a high proportion TATA-less promoters contain CREB binding sites, these promoters appear to be generally unresponsive to cAMP, suggesting activation of these promoters depends on co-stimulatory signals. In this study we observed co-activation of the CH25H promoter by Sp1 and CREB in a synergistic manner. A negative feedback mechanism involving 25HC was also observed in the in vitro reporter assays and this effect appeared to be mediated by an element within the proximal 250 bp of the promoter. The region containing the p53 consensus site was demonstrated to repress transcription.