Abstract: Protoplasts were isolated from the mycorrhizal fungus Cenococcum geophilum. To obtain high yield of protoplast,it was very effective to pretreat the mycelium with trypsin(20μg·mL-1 for 1h)and tlien incubated the mycelium in the mixture containing the lytic enzymes with 0.8M sorbitol as the osmotic stabilizer. The effects of different combination of lytic enzymes,density of mycelium in incubation and incubation temperature on the yield of protoplast were investigated.In MMNSO and MMNKCl solid(2%agar)or liquid medium,the protoplasts were regenerated and reversed into normal hyphae. Regeneration rate of protoplast was about 1%～5%.Test of specific activities of glucose-6-phosphate dehydrogenase and mannitol dehydrogenase indicated that there was a similar activity in protoplast as compared with that in mycelium.