[Background] Based on the sequence analysis of a novel recombinant highly thermostable b-mannanase (ReTMan26) from a thermophilic Bacillus subtilis (TBS2), there are 3 N-glycosylation sites (N8, N26 and N255) in the encoding gene of ReTMan26, and ReTMan26 could be N-glycosylated when expressed by Pichia pastoris. [Objective] To determine the effects of N-glycosylation on the stability of ReTMan26. [Methods] Through constructing the three-dimensional structure models, the effects of N-glycosylation on the stability of ReTMan26 were analyzed. Then, the N-deglycosylated ReTMan26 (ReTMan26-DG) was obtained using Native Protein Deglycosylation Kit. After purification, the differences of enzymatic stability between ReTMan26 and ReTMan26-DG were determined. [Results] The optimum reaction pH of ReTMan26 was 6.0, identical with that of ReTMan26-DG, and pH stability of ReTMan26 was slightly higher than that of ReTMan26-DG in pH range between 1.5 and 9.0. The optimum temperature of ReTMan26 was 60 °C, 5 °C higher than that of ReTMan26-DG. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min. However, ReTMan26-DG retained 58.2% residual activity after treatment at 93 °C and was completely inactivated after treatment at 100 °C for 10 min. After treatment with trypsin or pepsin at 37 °C for 2 h, ReTMan26 retained 91.2% and 70.5% of its baseline activity, 23.7% and 25.6% higher than ReTMan26-DG, respectively. [Conclusion] N-glycosylation could improve the stability of ReTMan26 at different pH, high-temperature and the resistance to digestive proteases.