Bifidobacterium dominate the colonic environment when infants are breast fed. Human milk contains high concentrations of human milk oligosaccharides that promote the growth of Bifidobacterium in a widely researched phenomenon. However, human milk has many bioactive components, including N-glycosylated lactoferrin and immunoglobulins, and there is evidence that species of Bifidobacterium also use these as a source of nutrition. Furthermore, being able to breakdown N-glycans is reported to be a strong colonisation factor for these microbes. To assess the ability of species of Bifidobacterium to access the sugars in N-glycans, bioinformatics, microbiology, biochemical, structural and glycobiology techniques were applied. Here, we describe the characterisation of eight distinct keystone enzymes from species of Bifidobacterium that remove N-glycans from protein. We used a variety of substrates to determine the specificity of these enzymes to understand the biology of how Bifidobacterium breakdown N-glycans. This work includes the first structure of the PNGaseA superfamily.