The human gut symbiontRuminococcus gnavusdisplays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 fromR.gnavusATCC 29149,RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS thatRgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmedRgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis ofRgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures ofRgGH98 in complex with BgA trisaccharide (BgAtri) and ofRgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally thatRgGH98 is part of an operon of 10 genes that is overexpresssed in vitro whenR.gnavusATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmedRgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed thatRgGH98 releases BgAtri from mucin and that pretreatment of mucin withRgGH98 conferedR.gnavusE1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire ofR.gnavusstrains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.