Abstract: Shikimate kinase is a key protein of the shikimic pathway, which is essential for the survival of Mycobacterium tuberculosis. In this study, a screening assay for Mycobacterium tuberculosis shikimate ki-nase (MtSK) inhibitor was developed. A 120 000-compound library was screened by the enzyme assay and the phenotype screening using Mycobacterium smegmatis. A hit compound named IMB-T5297(E)-3-(3-(3- chloro-5-methoxy-4-(prop-2-yn-1-yloxy)phenyl)acryloyl)-6-methyl-2H-pyran-2,4(3H)-dione was identified to be a selective inhibitor of MtSK with a half maximal inhibitory concentration (IC₅₀) value of 1.745 μg·mL^-1, which also showed antibacterial activity. The interaction between compound and protein was analyzed by surface plasmon resonance (SPR) experiment, which showed the K_D value was 2.151×10^-5 mol·L^-1. The binding model of MtSK and compound was simulated by the computer program. Five key amino acids in the binding pocket were indispensable site-directed mutated to verify the model. IMB-T5297 inhibited Mycobacterium tuberculosis H37Rv with a minimum inhibitory concentration (MIC) value of 49.723 μg·mL^-1 and displayed low cytotoxicity to mammalian cells. In this study, IMB-T5297 was identified as a selective inhibitor of MtSK enzyme with anti-tuberculosis activity. With additional structural modification, the compound has a potential to become a novel anti-tuberculosis compound.