We here demonstrate that promoter polymorphisms rs8003602 and rs3783320 of cholesterol 24S-hydroxylase (CYP46A1) were significantly associated with Alzheimer's disease (AD) in Chinese subjects. Haplotype analyses showed that haplotype CG is the risk haplotype. Either single marker or haplotypic association was found only in the APOE ε4 negative group. The association was then replicated in an independent set of case-control samples in Mongolians. We also investigated the function of promoter haplotypes and found that luciferase expression for TA promoter construct exhibited significantly higher expression level than the risk CG promoter construct. This finding might indicate individuals bearing the CG haplotype are genetically more susceptible to AD compared to those with TA haplotype. Further, we found MYT1 could be the potential transcription factor binding to the significant promoter polymorphism and mediated gene transcriptional activity. In general, our results show that promoter haplotypes could significantly affect CYP46A1 gene transcription level possibly through interacting with certain transcription factors.