Aims: Improvement of cellulase production of Penicillium decumbens by genome shuffling of an industrial catabolite-repression-resistant strain JU-A10 with its mutants. Methods and Results: After two rounds of genome shuffling, three fusants, GS2-15, GS2-21 and GS2-22, were obtained, showing 100%, 109% and 94% increase in FPase activity than JU-A10 respectively. The cellulase production of the fusants on various substrates, such as corn stover, wheat straw, bagasse and the corncob residue, was studied. The maximum productivities of GS2-15, GS2-21 and GS2-22 were 92 center dot 15, 102 center dot 63 and 92 center dot 35 FPU l-1 h-1 on the corncob residue at 44 h respectively, which were 117%, 142% and 118% higher than that of JU-A10 (42 center dot 44 FPU l-1 h-1, at 90 h). The improvements of the fusants were possibly because of their enhanced growth rates, earlier cellulase synthesis and higher secretion of extracellular proteins. Conclusions: The fusants obtained after genome shuffling could produce abundant cellulase much earlier, and they could be potential candidates for bioconversion process. Significance and Impact of the Study: This is the first report on the improvement of cellulase production in fungi by genome shuffling, and this is a good technique to improve other important phenotypes in fungi.