Objectives: Drug-resistant Mycobacterium tuberculosis poses a great threat to human health.Tyrosyl-tRNA synthetase(TyrRS)is one of the aminoacyl tRNA synthetases that catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis.There are several distinctive differences between bacterial and human TyrRS and therefore it could be a potential target for developing antimicrobial agents.This study aimed to identify a new anti-TB agent targeting M.tuberculosis TyrRS(MtTyrRS).Methods: We first used Mycobacterium smegmatis for a phenotypic screening of 20000 compounds.The hit compounds were then screened with MtTyrRS.The interaction between hit compound IMB-T130 and the target protein was analysed by surface piasmon resonance(SPR)assay and molecular docking experiments.The target of IMB-T130 was further confirmed by the overexpression of the target protein.The antibacterial activity of IMB-T130 against various standard and clinical drug-resistant M,tuberculosis strains was evaluated using the microplate Alamar blue assay.Results: Compound IMB-T130 was identified as a hit compound that inhibits the growth of M.smegmatis and the in vitro activity of MtTyrRS.The interaction between IMB-T130 and MtTyrRS was confirmed by SPR assay and molecular docking analysis.The higher MIC for a strain overexpressing the target protein also suggests that MtTyrRS is likely to be the target of IMB-T130.IMB-T130 shows excellent anti-TB activity and low cytotoxicity.Conclusions: IMB-T130 inhibits the growth of MDR-TB and XOR-TB by targeting MtTyrRS.Because of its low cytotoxicity against mammalian cells,IMB-T130 is a promising new agent against drug-resistant M.tuberculosis.