Intestinal mucous layers mediate symbiosis and dysbiosis of host–microbe interactions. These interactions are influenced by the mucinO-glycan degrading ability of several gut microbes. The identities and prevalence of many glycoside hydrolases (GHs) involved in microbial mucinO-glycan breakdown have been previously reported; however, the exact mechanisms and extent to which these GHs are dedicated to mucinO-glycan degradation pathways warrant further research. Here, usingBifidobacterium bifidumas a model mucinolytic bacterium, we revealed that two β-N-acetylglucosaminidases belonging to the GH20 (BbhI) and GH84 (BbhIV) families play important roles in mucinO-glycan degradation. Using substrate specificity analysis of natural oligosaccharides andO-glycomic analysis of porcine gastric mucin (PGM) incubated with purified enzymes orB. bifidumcarryingbbhIand/orbbhIVmutations, we showed that BbhI and BbhIV are highly specific for β-(1→3)- and β-(1→6)-GlcNAc linkages of mucin core structures, respectively. Interestingly, we found that efficient hydrolysis of the β-(1→3)-linkage by BbhI of the mucin core 4 structure [GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα-O-Thr] required prior removal of the β-(1→6)-GlcNAc linkage by BbhIV. Consistent with this, inactivation ofbbhIVmarkedly decreased the ability ofB. bifidumto release GlcNAc from PGM. When combined with abbhImutation, we observed that the growth of the strain on PGM was reduced. Finally, phylogenetic analysis suggests that GH84 members may have gained diversified functions through microbe–microbe and host–microbe horizontal gene transfer events. Taken together, these data strongly suggest the involvement of GH84 family members in host glycan breakdown.