Abstract: In order to investigate the purification and its enzymatic characteristics of N-acetyl-β-D-glucosaminidase (EC3.2.1.52, NAGase) from intestinal tract of Japanese eel, Anguilla japonica, NAGase was purified by extraction with ammonium sulfate fractionation, then chromatographed on Sephadex G-100 followed by DEAE-cellulose (DE-32) columns. The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The kinetic parameters of NAGase for the hydrolysis of pNP-β-D-GlcNAc (enzyme substrate) and enzymatic characteristics were also determined. The specific activity of the purified enzyme was 2517.40 U/mg. The molecular weight of enzyme was 69.98 kD. The optimum pH and optimum temperature of the enzyme were 6.0 and 60℃, respectively. The K_m value was 0.336 mmol/L and the V_max value was 7.634 µmol/(L·min), respectively. The enzyme was stable with pH of 4.8 to 7.2 and temperature of 4—60℃. The enzyme lost its activity rapidly when temperature >65℃. The effects of metal ions on the enzyme were also studied. Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺ and Fe³⁺ showed different degrees of activation effects on the NAGase. Na⁺, Li⁺ and Ba²⁺ had no influence on enzyme activity. Zn²⁺, Fe²⁺, Pb²⁺ and Hg²⁺ showed various degrees of inhibitory effects on the NAGase. Hg²⁺ inhibited the enzyme the most, and the enzyme activity decreased by 83.69% when its concentration reached 1.0 µmol/L. The essential groups of the NAGase were investigated using chemical modification method. The results demonstrated that essential groups of NAGase included lysine's ԑ-amidogen group, cysteine's sulfhydryl group, histidine's imidazolyl group, serine's hydroxyl group and tryptophan's indole group, while guanidyl of arginine was not an essential group of enzyme. Disulfide bond was essential for the catalytic activity of the enzyme. In conclusion, the purification scheme of NAGase from intestine of Anguilla japonica was effective and feasible. The activity of enzyme was affected easily by acidity-alkalinity, temperature and metal ions. The enzyme had similar essential groups to the NAGase from other animal sources.