N-glycosylation is an important post-translational modification of proteins in all eukaryotes and involved in a number of diseases in mammalian systems. However, little is known about the role of protein N-glycosylation in plant defense responses to pathogen invasion. In the present study, we first identified glycoproteins related to systemic acquired resistance (SAR) in an Arabidopsis thaliana model using glycoproteomics platform based on high-resolution mass spectrometry. In total, 427 glycosylate sites corresponding to 391 glycopeptides and 273 unique glycoproteins were identified. A total of 65 significantly changed glycoproteins with 80 N-glycosylation were detected in systemic leaves of SAR-induced plants, including numerous GDSL-like lipases, thioglucoside glucohydrolases, kinases and glycosidases. A variety of significantly changed glycoproteins were involved in stomatal movement, and stomata aperture measurements further confirmed that stomata movement were regulated in systemic leaves of SAR-induced plants, suggesting that these proteins may be functionally involved in systemic stomatal immunity through glycosylation or deglycosylation. Functional enrichment analysis reveals that the significantly changed glycoproteins were mainly involved in N-glycan biosynthesis and degradation, phenylpropanoid biosynthesis, cutin and wax biosynthesis, plant-pathogen interactions. Comparative analysis of glycoproteomics data with proteomics and transcriptomics data suggest that these significantly changed glycoproteins were mainly regulated by post-translational modification during SAR. This study provides substantial insight into the role of protein glycosylation in SAR.