The released milk N-glycome has been found to possess antipathogenic activity. Natively, they are covalently linked onto proteins. Whether the conjugated N-glycans still have antipathogenic properties and how the glycosylation influences the antipathogenic activity of proteins remain unclear. Herein, we compared the quantitative differences of milk protein N-glycosylation and the antilisterial differences of native milk proteins, released N-glycan pools, and deglycosylated proteins between human and bovine milk. N-glycosylation exhibited to be quantitatively species-specific. The entire growth inhibitory activity and the majority of the antiadhesive activity against Listeria monocytogenes of milk whey proteins, although not as high as the released N-glycans, are attributed to N-glycosylation. Moreover, all N-glycan-bearing samples from human milk showed better growth inhibitory activities than those from bovine milk. Generally, N-glycosylation significantly contributes to the antilisterial function of milk proteins and to the functional differences between species. This gives novel insights into the role of these glycoconjugates in nature.