A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference. (C) 2012 Elsevier Ltd. All rights reserved.