Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. Here, EndoBI-1 was used to release these N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis, a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacteriumanimalis subsp. lactis, a species which does not. Conversely Bifidobacterium longum subsp. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction clearly demonstrating that the glycan portion of milk glycoproteins provides the key substrate for growth. Mass spectrometry-based profiling revealed that B.longum subsp. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B.animalis subsp. lactis only degraded 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-borne bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans are better growth substrates than the intact milk glycoproteins suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides.