Transgenic silkworm expression systems have been applied for producing various recom- binant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm’s genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silk- worms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials