IMPORTANCE The use of anticholinergic (AC) medication is linked to cognitive impairment and an increased risk of dementia. To our knowledge, this is the first study to investigate the association between AC medication use and neuroimaging biomarkers of brain metabolism and atrophy as a proxy for understanding the underlying biology of the clinical effects of AC medications. OBJECTIVE To assess the association between AC medication use and cognition, glucose metabolism, and brain atrophy in cognitively normal older adults from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and the Indiana Memory and Aging Study (IMAS). DESIGN, SETTING, AND PARTICIPANTS The ADNI and IMAS are longitudinal studies with cognitive, neuroimaging, and other data collected at regular intervals in clinical and academic research settings. For the participants in the ADNI, visits are repeated 3, 6, and 12 months after the baseline visit and then annually. For the participants in the IMAS, visits are repeated every 18 months after the baseline visit (402 cognitively normal older adults in the ADNI and 49 cognitively normal older adults in the IMAS were included in the present analysis). Participants were either taking (hereafter referred to as the AC+ participants [52 from the ADNI and 8 from the IMAS]) or not taking (hereafter referred to as the AC- participants [350 from the ADNI and 41 from the IMAS]) at least 1 medication with medium or high AC activity. Data analysis for this study was performed in November 2015. MAIN OUTCOMES AND MEASURES Cognitive scores, mean fludeoxyglucose F 18 standardized uptake value ratio (participants from the ADNI only), and brain atrophy measures from structural magnetic resonance imaging were compared between AC+ participants and AC- participants after adjusting for potential confounders. The total AC burden score was calculated and was related to target measures. The association of AC use and longitudinal clinical decline (mean [SD] follow-up period, 32.1 [24.7] months [range, 6-108 months]) was examined using Cox regression. RESULTS The 52 AC+ participants (mean [SD] age, 73.3 [6.6] years) from the ADNI showed lower mean scores onWeschler Memory Scale-Revised Logical Memory Immediate Recall (raw mean scores: 13.27 for AC+ participants and 14.16 for AC- participants; P = .04) and the Trail Making Test Part B (raw mean scores: 97.85 seconds for AC+ participants and 82.61 seconds for AC- participants; P = .04) and a lower executive function composite score (raw mean scores: 0.58 for AC+ participants and 0.78 for AC- participants; P = .04) than the 350 AC- participants (mean [SD] age, 73.3 [5.8] years) from the ADNI. Reduced total cortical volume and temporal lobe cortical thickness and greater lateral ventricle and inferior lateral ventricle volumes were seen in the AC+ participants relative to the AC- participants. CONCLUSIONS AND RELEVANCE The use of AC medication was associated with increased brain atrophy and dysfunction and clinical decline. Thus, use of AC medication among older adults should likely be discouraged if alternative therapies are available.