A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11TS. AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (Topt) of AEx11A was 75 ℃ and its half-life at 70 ℃ (t1/270) was 197 min, improved as compared with those (Topt = 50 ℃, t1/270 = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A’s structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5–Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the Topt of the mutant AEx11A (AEx11AC5T) dropped to 60 ℃ from 75 ℃ of AEx11A, and its t1/270 and t1/280 also decreased to 3.0 and 1.0 min from 197 and 25 min.