Locoweeds are toxic plants of the genera Astragalus and Oxytropis containing swainsonine.Swainsonine is the principal agent responsible for locoism in animals.Recently, the fungal endophytes are isolated frequently from locoweeds, Undifilum spp, are believed that responsible for the production of swanisonine in locoweeds.Currently,the mechanism was few known about the biosynthesis of swainsonine in Locoweeds endophytic fungi.The swainsonine-pruducing endophytic fungi, Undifilum oxytropis, was used to develop protoplasts in this research.This study will provide a important basis for further to elucidate the swainsonine biosynthetic pathways and build a non-toxic fungus.The effects of some factors on formation and regeneration of protoplasts were investigated, including culture method, mycelium age, digesting time and temperature.The results showed that the highest preparation rate was achieved when prepared through mixture solution of 1% cellulase + 1% snailase + 0.1% lysing enzyme, at 35 ℃, 80 r/min for 3 h.The regeneration colonies were achieved when cultured in YCM medium for 9 d at 22 ℃ and the highest regeneration rate (8.5 %) was achieved when cultured in YCM medium for 15 d at 22 ℃.The color of regeneration colonies was different from wild strain and the form of regeneration mycelium was the same as wild strain when cultured in YCM medium.The regeneration mycelium could still produce swainsonine.The preparation and regeneration of protoplasts of the swainsonine-producting endophytic fungi, Undifilum oxytropis, which would providing important tool for the research of genetic transformation and genetic manipulation and science basis for further to elucidate the synthesis of swainsonine in molecular level.