Brain region-specific deposition of extracellular amyloid plaques principally composed of aggregated amyloid-β (Aβ) peptide is a pathological signature of Alzheimer’s disease (AD). Recent human neuroimaging data suggest that resting-state functional connectivity strength is reduced in patients with AD, cognitively normal elderly harboring elevated amyloid burden, and in advanced aging. Interestingly, there exists a striking spatial correlation between functional connectivity strength in cognitively normal adults and the location of Aβ plaque deposition in AD. However, technical limitations have heretofore precluded examination of the relationship between functional connectivity, Aβ deposition, and normal aging in mouse models. Using a novel functional connectivity optical intrinsic signal (fcOIS) imaging technique, we demonstrate that Aβ deposition is associated with significantly reduced bilateral functional connectivity in multiple brain regions of older APP/PS1 transgenic mice. The amount of Aβ deposition in each brain region was associated with the degree of local, age-related bilateral functional connectivity decline. Normal aging was associated with reduced bilateral functional connectivity specifically in retrosplenial cortex. Furthermore, we found that the magnitude of regional bilateral functional correlation in young APP/PS1 mice prior to Aβ plaque formation was proportional to the amount of region-specific plaque deposition seen later in older APP/PS1 mice. Together, these findings suggest that Aβ deposition and normal aging are associated with region-specific disruption of functional connectivity and that the magnitude of local bilateral functional connectivity predicts regional vulnerability to subsequent Aβ deposition in mouse brain.