In order to obtain high quality protoplasts for the transformation of Rhizoctonia solani Kühn AG-1 IA,isolate GD-118 of this pathogen was used for the protoplast preparation and regeneration by optimizing the conditions in six aspects for the preparation of protoplast, including enzymes, mycelial ages, digest times, digest temperature, digest pH and osmotic stabilizers and their concentrations, and in two aspects for the regeneration of protoplast, including digest time and osmotic stabilizers together with their concentrations. The results showed that the best optimized combination conditions for the preparation of protoplast were “cellulase + lysozyme + driselase” at final total enzyme concentration of 10 mg/mL, 15 h of mycelial age, 3 h of digestion, 0.6 mol/L of MgSO4·7H2O as osmotic stabilizer,35 ℃ of digest temperature and pH 5.6, which gave the protoplast yield as high as 4.00×107/g fresh mycelia; whereas 3 h of digestion and 1.0 mol/L of mannitol were the best optimized conditions for the regeneration of protoplast in this study, and the regeneration rate was 39%. 