Objective To explore the mechanismand possible drug target of the new anti-tuberculosis compound, S28, which is effective on Mycobacterium tuberculosis in vitro.Methods Two dimensional gel electrophoresis ( 2 - DE) was employed to determine the effects of this compound on the expression of Mycobacterium tuberculosis proteome. Global proteome of Mycobacterium tuberculosis strain H37Ra was treated with S28 and compared with control ( untreated) . Results The expression of 13 proteins was decreased; six proteins, which were remarkably decreased in Mycobacterium tuberculosis treated with S28, were subjected to matrix assisted laser adsorption/ionization time-of-flight mass spectrometry ( MALDI - TOF- MS) analysis and the nature of two proteins was successfully determined. They were identified as elongation factor Tu and short chain dehydrogenase, which play important roles in protein translation and energy metabolism. Conclusion These data provide insight into potential novel anti-tuberculosis mechanisms and drug targets.