Update publication information. Being part of the human diet, peach is an important fruit consumed worldwide. In the present study, a systematic first insight into the N-glycosylation of peach fruit during ripening was provided. First, N-glycome by reactive MALDI-TOF mass spectrometry indicated that six of 24 N-glycans of peach were differentially expressed. Second, a comparative N-glycoproteome was characterized via 18O-tagged N-glycosylation site labeling followed by nLC-ESI-MS/MS. Totally 1464 N-glycosites on 881 N-glycoproteins were identified, among which 291 N-glycosites on 237 N-glycoproteins were expressed differentially with a fold change value of 1.5 or 0.67. The enrichment analysis of GO and KEGG pathways revealed that four pathways including other glycan degradation, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and protein processing in endoplasmic reticulum were mainly enriched, in which several important N-glycoproteins with dynamic change during fruit ripening were further screened out. Our findings on a large scale for N-glycosylation analysis of peach during ripening may provide new molecular insights for comprehending N-glycoprotein functions which should be of great interest to both glycobiologists and analytical chemists.