Previous studies showed that suppression of N-glycan processing enzymes like β-N-acetylhexosaminidase could significantly enhance the fruit shelf life. It indicated that N-glycosylated proteins in the cell wall are also important targets involved in ripening and softening. Based on the techniques of qualitative and quantitative analysis of N-glycan, we explored the changing of N-glycans during the process of ripening and softening of peach fruits. It was founded that the proportion of N-glycans bearing terminal N-acetylglucosamine was significant decreased during the ripening while the activity and expression level of β-N-acetylhexosaminidase were increased. And the changing was also proved to be associated with the ethylene evolution and respiratory intensity. The evidences indicated that protein conjugated N-glycans might be involved in fruit ripening and softening of peach and regulated by ethylene signal pathway.