Abstract: The subcellular proteomics play an important role in study of minor proteins, which participating the intracellular metabolism. Subcellular proteomics can increase the minor proteins separation by twodimensional electrophoresis (2DE). A comparative analysis of proteomic profiling was conducted between nuclei of healthy mammary gland and infected clinical mastitis of dairy cow, to find more valuable biological molecular labels involved in mastitis pathogenesis. In this study, nuclei were isolated and purified by ultracentrifuge, and then twodimensional electrophoresis was applied to separate nuclear proteins. Imagine analysis for selection of differentiallyexpressed protein spots was conducted by PDQUest 7.4 software. By ion trap mass spectrometry equipped with surveyor HPLC system proteins were identified. 22 spots from electrophoresis gels were selected as differentiallyexpressed proteins for MS analysis and 17 proteins were identified. Seven of differentiallyexpressed proteins showed upregulated expression and 2 proteins showed downregulated expression during mastitis. 5 proteins only expressed in healthy mammary gland and 3 proteins only expressed in mammary gland which infected with clinical mastitis. The differentiallyexpressed proteins separated in this study had a wide range of functions which are related to the formation of cellular skeleton, metabolic regulation and apoptosis regulation. These suggested that nuclei of mammary gland had experienced fundamental changes in structure and metabolism when contracted clinical mastitis.