Protein modification by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer's disease (AD); however, detailed molecular characterization of this important protein post‐translational modification at the proteome level has been highly challenging, owing to its low stoichiometry and labile nature. Herein, we report the most comprehensive, quantitative proteomics analysis for protein O‐GlcNAcylation in postmortem human brain tissues with and without AD by the use of isobaric tandem mass tag labelling, chemoenzymatic photocleavage enrichment, and liquid chromatography coupled to mass spectrometry. A total of 1850 O‐GlcNAc peptides covering 1094 O‐GlcNAcylation sites were identified from 530 proteins in the human brain. One hundred and thirty‐one O‐GlcNAc peptides covering 81 proteins were altered in AD brains as compared with controls (q < 0.05). Moreover, alteration of O‐GlcNAc peptide abundance could be attributed more to O‐GlcNAcylation level than to protein level changes. The altered O‐GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory‐associated proteins. These findings suggest that dysregulation of O‐GlcNAcylation of multiple brain proteins may be involved in the development of sporadic AD. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.