Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.