Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases worldwide, with no effective treatments or preventions. Evidence suggests that environmental factors, including dietary nutrients, contribute to the etiology of AD. Choline is an essential nutrient found in many common foods. Choline is produced endogenously, but not at levels sufficient for healthy metabolic function and thus requires dietary supplementation. Literature shows that ~90% of Americans do not meet the adequate intake threshold for dietary choline consumption and therefore are dietary choline-deficient. While dietary choline supplementation throughout life has been shown to have significant health benefits, such as reducing AD pathology and improving cognition in a mouse model of AD, the impacts of dietary choline deficiency are unknown. Experiments were designed to understand the effects of dietary choline deficiency in healthy, non-transgenic mice (NonTg) and in the 3xTg-AD mouse model of AD. From 3 to 12 months of age, mice received either adequate choline (ChN) in the diet or were put on a choline-deficient (Ch-) diet. A Ch- diet leads to significant weight gain throughout life in both the NonTg and 3xTg-AD mice, with AD mice showing a greater increase. Additionally, impaired glucose metabolism, which is a risk factor for AD, was induced in both NonTg Ch- and 3xTg-AD Ch- mice. Interestingly, Ch- induced cardiomegaly in 3xTg-AD mice and elevated markers of cardiac dysfunction in NonTg mice to similar levels in 3xTg-AD mice. Finally, Ch- exacerbated amyloid-β plaque pathology and tau hyperphosphorylation in the hippocampus and cortex of 3xTg-AD mice. Proteomic analyses revealed Ch- induced changes in hippocampal proteins associated with postsynaptic receptor regulation, microtubule stabilization, and neuronal development, as well as well-known AD-associated proteins (MAPT, BACE1, MECP2, CREBBP). Proteomic analyses also revealed Ch- induced changes of plasma proteins associated with secondary pathologies of AD including inflammation, immune response insulin metabolism, and mitochondrial dysfunction (SAA1, SAA2, IDE, HSPD1, VDAC-1, VDACE-2). Taken together, these data suggest that dietary choline deficiency induces system-wide cellular and molecular dysfunction associated with AD across several pathogenic axes, through proteomic changes not only in the hippocampus but also in the plasma.