Objective To explore the optimal methods for the protoplast preparation and regeneration ofHirsutella sinensisby optimizing the limiting factors. Results During the treatment of enzymatic protoplast preparation, mycelium cultured for 7 days was the optimal start material. The maximum protoplast preparation rate of 4.3 x 10(7)protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH2PO4-K(2)HPO(4)buffer (pH 5.5) with 0.6 M KCl at 18 degrees C. As for the protoplast regeneration, the maximum protoplast regeneration rate reached 12.32% through 5 x 10(3)protoplasts mL(-1)cultivated for 20 days in the regeneration medium with 0.6 M mannitol and 1.5% agar. Conclusions The preparation and regeneration ofH. sinensisprotoplasts was firstly established based on process optimization and it provided a foundation for the study ofH. sinensismutagenesis.