This study was aimed to perform a comparative analysis on potential secretory proteins between high (HB0801) and low (P150) virulent M.bovis strains. We employed the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based label-free quantitative proteomic technology to identify and characterize the differential secretome profiles for two M.bovis strains. A total of 477 secretory proteins were identified in both two strains and 165 deferentially expressed in HB0801 over 1.5-folds higher than P150.