Complex, indigestible free oligosaccharides as well as conjugated glycans are found in milk that shape the gut microbiome of infants. The activity of an endo-beta-N-acetylglucosaminidase from B. longum subsp. infantis (B. infantis) is known to release N-glycans from native milk glycoproteins under physiological conditions. We investigated whether this enzyme is active in vivo in breastfed infants fed B. infantis EVC001. Using mass spectrometry, we found 19 N-glycans related to human milk glycoproteins increased in abundance, similar to previous work using bovine milk glycoproteins, and these 19 N-glycans matched unique specificities of this enzyme. Twenty N-glycans were unique to infants fed B. infantis EVC001. Bifidobacteriaceae were correlated with these glycans, confirming the relationship between B. infantis and released N-glycans. This suggests that this enzyme is active in vivo and releases N-glycans from milk glycoproteins, and may play a role in B. infantis EVC001 colonization of the gut microbiome.