Xylanase is one of the most important hemicellulases in industry. However, its low thermostability limits its applications. In this study, one thermostable xylanase-producing strain 400264 was obtained from screening 11 Aspergillus niger strains (producing thermotolerant xylanase), and the optimum temperature of crude xylanase extracted from it was 55°C. Original activity of the crude xylanase is 64% at 60°C and 55% at 85°C with an incubation time of 30 min, respectively. After the expression of recombinant xylanase gene (xynA/xynB), the XYNB (xylanase B) showed higher thermostability than XYNA (xylanase A). Recombinant enzyme XYNB retained 94% of its activity for 10 min at 85°C, while XYNA with no activity left. Site-directed mutagenesis was performed to replace Ala33 of XYNB by Ser33 resulting 19% decrease in enzyme activity after incubating at 85°C for 30 min. It suggested that the Ala33 residue may have a certain effect on the thermophilic adaptation of xylanase.