Cyathus stercoreus has a superior ability to increase the cellulase saccharification yields of rice straw under biological pretreatment. As a first step to improve the biological pretreatment efficiency of C. stercoreus further, a new gene transformation system was constructed. Uracil auxotrophic mutant uv-180 was generated as host strain by ultraviolet radiation. It was transformed using plasmid pGE containing the orotate phosphoribosyltransferase (CsURA5) and enhanced green fluorescent protein (egfp). Many transformants exhibited strong fluorescence, indicating successful genetic transformation. An intron was needed for the expression of egfp. The EGFP fluorescence intensities of the three transformants did not decay even after subculturing on uracil-containing semisolid medium 5 times, suggesting that the transformed plasmids were stably retained in the absence of selective pressure.