CpGH89 is a family 89 glycoside hydrolase with exo-α-D-N-acetylglucosaminidase activity that is produced by the human and animal pathogen Clostridium perfringens. This enzyme is active on the α-D-GlcpNAc-(1 → 4)-D-Galp motif that is displayed on the class III mucins within the gastric mucosa. Other members of this enzyme family, such as human NAGLU, are active on heparan. A truncated version of CpGH89 was rendered inactive through the mutation of two key catalytic residues, the protein crystallized and its structure determined in complex with α-D-GlcpNAc-(1 → 4)-D-Galp to reveal the molecular details of how this unique disaccharide is recognized by CpGH89. An analysis of this substrate complex not only provides insight into how this enzyme selects for its mucin-presented substrate but also advances our understanding of how its clinically relevant mammalian counterparts are specific for heparan.