The filamentous Ascomycete fungus Undifilum oxytropis is an endophyte of locoweed plants of the genera Oxytropis that produces a toxic alkaloid swainsonine. Swainsonine, an alpha-mannosidase inhibitor causes a general toxicosis and neurological problems (locoism) when consumed by grazing animals. Swainsonine is also being assessed for its anti-cancer properties. While the ecology of U. oxytropis has been studied, little is known about the genetics and proteomics of any swainsonine-producing fungus. To help understand the proteins in U. oxytropis , the proteome of U. oxytropis was analyzed using 2-dimensional electrophoresis (2-DE). Proteins from U. oxytropis mycelia were extracted and separated by in-gel isoelectric focusing (IEF). The entire immobilized pH gradient (IPG) strip was cut into a set of gel sections and each gel section was digested with trypsin and then identified using liquid chromatography tandem mass spectrometry (LC-MS/MS). 2-DE maps were also developed for U. oxytropis to define its proteome. In the isoelectric point (pI) range of 3-11 and 10-250 kDa ranges, more than 450 spots were detected in 2-DE silver-stained gels, and 52 proteins were identified by LC-MS/MS. Most of the identified proteins were involved in energy production, oxidoreductase activity, carbohydrate metabolic process, amino acid and cellular ketone metabolic process. A large group of identified proteins were related to stress proteins and heat shock proteins. This work presents the first two-dimensional reference map of this alkaloid-producing fungus. Details of the proteome serve as a baseline for further study of this swainsonine-producing fungus and are essential for a reverse genetic analysis of the fungus. Keywords: Undifilum oxytropis fungus, two-dimensional gel electrophoresis, proteome reference map, liquid chromatography tandem mass spectrometry, swainsonine