Abstract: Effect of regenerating method,osmotic pressure stabilizer,culture medium,age of mushroom,etc.on the regeneration of Pleurotus eryngii protoplast was investigated.The results showed that the protoplasts from the 5-7days old mycelia yielded the most desirable results when cultured in a yeast peptone regeneration medium with up to 0.9% of NaCl as the osmotic stabilizer.Lowered temperature,shortened time and reduced enzyme concentration could help minimize enzymatic hydrolysis in the preparation which would benefit the protoplast regeneration.