Proteomics has become a powerful technology being successfully used in plant science research to analyze pathogen pathogenicity.Two-dimensional electrophoresis (2-DE) is a potential analytical tool in proteomics to identify qualitative and quantitative changes of proteins and reveal the changes of protein expression under different growth conditions or different test materials.However, protein sample preparation is a key fundamental step to 2-DE analysis, and it is also a prerequisite for proteomics analysis.X900 is a nonpathogenic mutant got from the wild type 03-8 in Valsa mali, but the wild type 03-8 is typical characterized with strong pathogenicity for apple tree.In order to efficiently analyze the differential expression proteins between the mutant x900 and its wild type 03-8, two mycelia protein extraction methods and three extracellular protein extraction methods were applied.