Cerebral deposition of the amyloid β protein (Aβ), an invariant feature of Alzheimer's disease, reflects an imbalance between the rates of Aβ production and clearance. The causes of Aβ elevation in the common late-onset form of Alzheimer's disease (LOAD) are largely unknown. There is evidence that the Aβ-degrading protease neprilysin (NEP) is down-regulated in normal aging and LOAD. We asked whether a decrease in endogenous NEP levels can prolong the half-life of Aβ in vivo and promote development of the classic amyloid neuropathology of Alzheimer's disease. We examined the brains and plasma of young and old mice expressing relatively low levels of human amyloid precursor protein and having one or both NEP genes silenced. NEP loss of function 1) elevated whole-brain and plasma levels of human Aβ40 and Aβ42, 2) prolonged the half-life of soluble Aβ in brain interstitial fluid of awake animals, 3) raised the concentration of Aβ dimers, 4) markedly increased hippocampal amyloid plaque burden, and 5) led to the development of amyloid angiopathy. A ∼50% reduction in NEP levels, similar to that reported in some LOAD brains, was sufficient to increase amyloid neuropathology. These findings demonstrate an important role for proteolysis in determining the levels of Aβ and Aβ-associated neuropathology in vivo and support the hypothesis that primary defects in Aβ clearance can cause or contribute to LOAD pathogenesis.