Alzheimer's disease (AD) is one of the most common neurodegenerative diseases that currently lacks clear pathogenesis and effective treatment.1-4. Protein glycosylation is ubiquitous in brain tissue and is closely related to important functions of the nervous system such as cognition and memory. Site-specific analysis of N-glycoproteome, which is technically challenging, can advance our understanding of the glycoproteins’ role in AD. To circumvent the challenges, our lab developed a large scale and precision identification method for site-specific N-glycopeptides named pGlyco 2.05. In this study, we profiled the multilayered variations in proteins, N-glycosites, N-glycans, and in particular site-specific N-glycopeptides in the APP/PS1 and wild type mouse brain through combining pGlyco 2.0 with other quantitative N-glycoproteomic strategies. The comprehensive brain N-glycoproteome landscape was constructed, and rich details of the heterogeneous site-specific protein N-glycosylations were exhibited. In particular, we have found that the overall down-regulation of the N-glycosylation modification in AD, and proved that down-regulation of glycosylation modification causes hyper-excitation and apoptosis of neuronal cells, as well as mis-localization and dysfunction of important proteins such as NCAM-1. In general, our work offered a panoramic view of the N-glycoproteomes in Alzheimer’s disease and revealed that generally impaired N-glycosylation promotes Alzheimer’s disease progression. Meanwhile, this study demonstrates the important role that general regulation of glycosylation modifications may have crucial effects on biological and pathological systems of AD and will broad the way for AD therapy.