Simple SummaryThe aim of this work was to combine xylanase expression and broiler production. The xylanase (XynB) gene from Trichoderma reesei was optimized to increase its expression level in Pichia pastoris. The maximum activity of xylanase (optiXynB) secreted by P. pastoris pPICZaA-optiXynB was 1299 U/mL after 96 h induction. The recombinase was highly specific towards birchwood xylan, beechwood xylan, and oat-spelt xylan. Dietary 1000 and 1500 IU/kg optiXynB significantly increased (p < 0.05) final weight and body weight gain; dietary 500, 1000, and 1500 IU/kg optiXynB significantly increased (p < 0.05) pre-evisceration weight, dressed percentage, and eviscerated weight compared with the control group. Results suggested that the optiXynB from P. pastoris pPICZaA-optiXynB has great application in broiler production.The aim of the present study was to improve the expression level of Trichoderma reesei xylanase (XynB) in Pichia pastoris through a codon optimization strategy and evaluate its effects on the growth performance and carcass characteristics of broiler. According to the codon bias of Pichia genome, the XynB gene from T. reesei was optimized and synthesized by whole gene assembly to improve its expression level in P. pastoris. Approximately 180 target mutations were successfully introduced into natural XynB. The maximum activity of xylanase (optiXynB) secreted by P. pastoris pPICZaA-optiXynB was 1299 U/mL after 96 h induction. Purified recombinant optiXynB had the molecular weight of 24 kDa. The optiXynB presented highest activity in pH 5.0 and 50 °C. The recombinase was highly specific towards birchwood xylan, beechwood xylan, and oat-spelt xylan. In the broiler experiment, a total of 200 Arbor Acre broilers (one day old) were randomly allocated into four groups fed with basal diets containing 0 (control group), 500, 1000, and 1500 IU/kg optiXynB. Dietary 1000 and 1500 IU/kg optiXynB significantly increased (p < 0.05) final weight and body weight gain; dietary 500, 1000, and 1500 IU/kg optiXynB significantly increased (p < 0.05) pre-evisceration weight, dressed percentage, and eviscerated weight compared with the control group. Inclusion of optiXynB in broiler diets linearly increased final weight, body weight gain, breast muscle weight and leg muscle weight, but linearly decreased feed conversion rate (p < 0.05). Furthermore, inclusion of optiXynB in broiler diets linearly and quadratically increased pre-evisceration weight, dressed percentage, and eviscerated weight (p < 0.05). The recombinant optiXynB from P. pastoris pPICZaA-optiXynB was beneficial in improving growth performance and carcass characteristics of broilers.