Alzheimer’s disease (AD) is the most common cause of dementia. It is characterized by β-amyloid (Aβ) plaques, neurofibrillary tangles and the degeneration of specifically vulnerable brain neurons. We observed high expression of the cholesterol 25-hydroxylase <i>(CH25H)</i> gene in specifically vulnerable brain regions of AD patients. <i>CH25H</i> maps to a region within 10q23 that has been previously linked to sporadic AD. Sequencing of the 5′ region of <i>CH25H</i> revealed three common haplotypes, <i>CH25H</i>χ<i>2</i>, <i>CH25H</i>χ<i>3</i> and <i>CH25H</i>χ<i>4</i>; CSF levels of the cholesterol precursor lathosterol were higher in carriers of the <i>CH25H</i>χ<i>4 </i>haplotype. In 1,282 patients with AD and 1,312 healthy control subjects from five independent populations, a common variation in the vicinity of <i>CH25H</i> was significantly associated with the risk for sporadic AD (p = 0.006). Quantitative neuropathology of brains from elderly non-demented subjects showed brain Aβ deposits in carriers of <i>CH25H</i>χ<i>4</i> and <i>CH25H</i>χ<i>3</i> haplotypes, whereas no Aβ deposits were present in <i>CH25H</i>χ<i>2</i> carriers. Together, these results are compatible with a role of <i>CH25H</i>χ<i>4</i> as a putative susceptibility factor for sporadic AD; they may explain part of the linkage of chromosome 10 markers with sporadic AD, and they suggest the possibility that <i>CH25H</i> polymorphisms are associated with different rates of brain Aβ deposition.