The aim of this study was to isolate and characterise the enzyme ß-N-acetylglucosaminidase (β-NAGase) in rainbow trout and Siberian sturgeon milt, in order to find similarities or differences in its involvement in the fertilization process of these two species. While sturgeon sperm possess an acrosome, teleosts do not. Applying ion exchanging chromatography to β-NAGase of rainbow trout sperm, two peaks of β-NAGase activity were obtained from the plasma material and from the sperm. The molecular weight was 74 kDa for both milt plasma forms and 127 kDa for rainbow trout spermatozoa peak. Optimum pH of purified rainbow trout enzyme peaks ranged from 4.6 to 5.0. Michaelis–Menten constants were 11.59 × 10−4 and 11.48 × 10−4 m for I and II milt plasma peaks. Incubation of purified peaks I and II at 56°C resulted in a 45 to 50% decrease in activity of both forms. Km value for rainbow trout spermatozoa β-NAGase was 5.4 × 10−4 m. Enzyme incubation tests at high temperature proved this form of the enzyme to be most resistant to high temperatures, since its activity after 20 min of incubation at 56°C decreased only by 25%. The molecular weight of the enzyme originating from Siberian sturgeon milt plasma was about 265 and 113 kDa for I and II milt plasma peaks, respectively, and 271 kDa for the sperm extract. Optimum pH for all sturgeon purified peaks ranged from 3.8 to 5.0. The Km constant was 5.12 × 10−4, 5.28 × 10−4 and 5.0 × 10−4 m for I and II milt plasma and spermatozoa peaks, respectively. The loss of ß-NAGase activity at 56°C was 58% for both milt plasma peaks and 35% for the sturgeon spermatozoa peak. The kinetic parameters, especially thermostability of ß-NAGase was similar to the homologous enzymes observed in mammalian semen.