Following contradictory reports, the aim of this study was to apply our highly specific novel assay to delineate the relationship between vitamin D forms and Alzheimer’s disease. The study incorporated patients, both untreated and treated with acetylcholinesterase inhibitors, along with controls. Patients were grouped as A: untreated (n=26) and B: treated with donepezil, rivastigmine or galantamine (n=44). The study included a control Group (C, n=35) with no cognitive impairment. Cognitive function was assessed using the MMSE. Levels of vitamin D forms were measured using liquid chromatography-mass spectrometry (LC-MS/MS) and calcium measurements were conducted using inductively coupled plasma-mass spectrometry (ICP-MS). In the cohort studied, no relationship was observed between MMSE score, calcium and any form of vitamin D. The indisputable finding is that the level of 25hydroxyvitamin D2 (25OHD2) (3.165±6.352 nmol/L, p < 0.001) was significantly lower in the untreated Group (A) compared to the control and treated groups (7.932±9.196 and 12.138±15.682 nmol/L, respectively). In contrast, the levels of the primary forms, vitamin D2 and total vitamin D were the highest for the untreated group. Vitamin D levels, assessed as 25OHD are significantly lower in patients suffering from Alzheimer’s disease arising from extremely low levels of 25OHD2 along with low levels of 25OHD3. Treatment with acetylcholinesterase inhibitors reverses this deficit. Further research is warranted to delineate the mode of action of acetylcholinesterase inhibitors with respect to normalising 25OHD2 levels. These observations resulted in the hypothesis that along with the common functions of vitamin D, different forms have distinct roles in health and disease.