[Background] As a selective marker, carboxin has been used in plant and fungi transformation. [Objective] To construct the genetic transformation system of Lentinula edodes by using carboxin resistant gene as a selective marker. [Methods] The mycelia of L. edodes strain 411-4 were collected at the 4th day after cultivation and then digested by lywallzyme to obtain the protoplast. Sufficient protoplast was mixed with the plasmid pL-cbx DNA and the polyethylene glycol (PEG) solution for transformation. The mixture finally was spread on the regeneration selecting plate, and the generated colonies were picked up for further testing. [Results] When more than 108 protoplasts and 4 μg plasmid DNA were used, 40 transformants were obtained. After being subcultured on media under carboxin selective stress, and the PCR identification, 38 transformants were positive and stable, indicating that carboxin gene was integrated into the genome of L. edodes 411-4. [Conclusion] The PEG mediated transformation method was constructed by using L. edodes strain 411-4 and carboxin selective marker.