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Abstract Background Vitamin B1 (VB1) is a crucial dietary nutrient and essential cofactor for several key enzymes in the regulation of cellular and metabolic processes, and more importantly in the activation of immune system. To date, the precise role of VB1 in Mycobacterium tuberculosis remains to be fully understood. Results In this study, the transcriptional and metabolic profiles of VB1-treated Mycobacterium. bovis BCG were analyzed by RNA-sequencing and LC-MS (Liquid chromatography coupled to mass spectrometry). The selection of BCG strain was based on its common physiological features shared with M. tuberculosis. The results of cell growth assays demonstrated that VB1 inhibited the BCG growth rate in vitro. Transcriptomic analysis revealed that the expression levels of genes related to fatty acid metabolism, cholesterol metabolism, glycolipid catabolism, DNA replication, protein translation, cell division and cell wall formation were significantly downregulated in M. bovis BCG treated with VB1. In addition, the metabolomics LC-MS data indicated that most of the amino acids and adenosine diphosphate (ADP) were decreased in M. bovis BCG strain after VB1 treatment. Conclusions This study provides the molecular and metabolic bases to understand the impacts of VB1 on M.bovis BCG.