Endoinulinase from Arthrobacter sp. S37 (EnIAb), a member of family 32 glycosidase, catalyzes hydrolysis of inulin using two catalytic residues, a nucleophile and an acid/base catalyst, as indicated in the bell-shaped pH dependence profile. Sequence alignment of EnIAb with other members of the family followed by a site-directed mutagenesis and the pH dependence studies of mutants proposed a detailed catalytic mechanism. Complete loss of enzyme activity in D460A mutant and noticeable changes in the alkaline limb from 8.8 to 7.0 of pH dependence in D460E mutant revealed that Asp460 residue, a component of the fully conserved RDP (Asn-Asp-Pro) motif among family 32, elevates the pKa of the acid/base catalyst of EnIAb with electrostatic interaction. Together with observations that only a kcat not a Km value was reduced more than 100-fold in E323A, E519A, D460N and D460E mutants, the active site of the enzyme is proposed to be a triad of carboxylate groups Glu323 (nucleophile), Glu519 (acid/base catalyst) and Asp460. Various single residue and truncated mutants of the unique N-terminal domain (residue 1 ~ 240) suggested that the domain has dual roles as a carbohydrate-binding domain and a direct involvement in catalysis. Significant reductions in enzyme efficiency (kcat/Km) were observed in W75K, W141A, and W141K mutants,suggesting these highly conserved aromatic residues W75 and W141 play a key role in substrate binding. In addition, N-terminal truncated mutants Δ15, Δ45, Δ70 as well as Δ240 were almost inefficient in enzyme catalysis, necessitating the structural integrity of the Nterminal domain for catalysis. These results provide a model of the active site common to a family 32 and a unique functional role of the N-terminal domain in EnIAb.