Endo-β-N-acetylglucosaminidase H hydrolyzes the β-(1–4)-glycosidic link of the N,N′-diacetylchitobiose core of high-mannose and hybrid asparagine-linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild-type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the β-barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the β-barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate-binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.