Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-β-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.