This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei. Successful expression of a gene of interest in a heterologous host involves front-end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled-up fermentation. The thermophilic xylanase B (XynB) enzyme from the bacterium Dictyoglomus thermophilum discussed here can be easily purified by heat-precipitation from the culture supernatant of the mesophilic host. A functional XynB can also be produced in Escherichia coli, but at a lower yield compared to that obtained in T. reesei. The protocol provided here can be adapted to various other proteins and filamentous fungal hosts. © 2018 by John Wiley & Sons, Inc.