Alzheimer's disease (AD) is pathologically characterized by tau-laden neurofibrillary tangles and β-amyloid deposits. Dysregulation of cholinergic neurotransmission has been implicated in AD pathogenesis, contributing to the associated memory impairments; yet, the exact mechanisms remain to be defined. Activating the muscarinic acetylcholine M₁ receptors (M₁Rs) reduces AD-like pathological features and enhances cognition in AD transgenic models. To elucidate the molecular mechanisms by which M₁Rs affect AD pathophysiological features, we crossed the 3xTgAD and transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant amyloid precursor protein (Tg-SwDI), two widely used animal models, with the M₁R^−/− mice. Our data show that M₁R deletion in the 3xTgAD and Tg-SwDI mice exacerbates the cognitive impairment through mechanisms dependent on the transcriptional dysregulation of genes required for memory and through acceleration of AD-related synaptotoxicity. Ablating the M₁R increased plaque and tangle levels in the brains of 3xTgAD mice and elevated cerebrovascular deposition of fibrillar Aβ in Tg-SwDI mice. Notably, tau hyperphosphorylation and potentiation of amyloidogenic processing in the mice with AD lacking M₁R were attributed to changes in the glycogen synthase kinase 3β and protein kinase C activities. Finally, deleting the M₁R increased the astrocytic and microglial response associated with Aβ plaques. Our data highlight the significant role that disrupting the M₁R plays in exacerbating AD-related cognitive decline and pathological features and provide critical preclinical evidence to justify further development and evaluation of selective M₁R agonists for treating AD.