Xylanases belonging to glycoside hydrolase (GH) family 11 have a wide range of pH optima. A single residue, which is located adjacent to the acid/base catalyst, is primarily responsible for pH optimum determination. This residue is Asp in acidophilic xylanases, whereas it is Asn in neutrophilic and alkaliphilic ones. Aspergillus kawachii produces 2 GH11 xylanases, acidophilic XynC, which has Asp37 at this position, and neutrophilic XynB, which has Asn43. To investigate the mechanism of pH optimum determination in these xylanases, we constructed various mutant enzymes, including mutations of the Asp/Asn residue. Their pH-dependent activities were measured using a natural xylan substrate or a synthetic substrate, o -nitrophenyl β-xylobioside. A D37N mutation raised the pH optimum of XynC from 2.8 to 5.5, whereas an N43D mutation lowered the pH optimum of XynB from 4.2 to 3.6. Crystallographic analysis on the D37N mutant of XynC suggested that a hydrogen bond between Asp(Asn)37 and the acid/base catalyst is weakened by the mutation. Kinetic analysis of the mutants suggested that the ionization states of the ES complex dictate the acidophilicity of XynC. Therefore, mutants of other residues in the active site cleft were also examined, and it was shown that Glu118 and Tyr10 also contribute to the extreme acidophilicity of XynC. Interestingly, an F131W mutation in XynC increased the activity toward the synthetic substrate by 8.4-fold. Crystallographic analysis on the F131W mutant suggested that optimization of the aromatic side chain packing in the substrate-binding cleft increases the catalytic activity.