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Tuberculosis remains a serious challenge to human health worldwide. <i>para</i>-Aminosalicylic acid (PAS) is an important anti-tuberculosis drug, which requires sequential activation by <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) dihydropteroate synthase and dihydrofolate synthase (DHFS, FolC). Previous studies showed that loss of function mutations of a thymidylate synthase coding gene <i>thyA</i> caused PAS resistance in <i>M. tuberculosis</i>, but the mechanism is unclear. Here we showed that deleting <i>thyA</i> in <i>M. tuberculosis</i> resulted in increased content of tetrahydrofolate (H₄PteGlu) in bacterial cells as they rely on the other thymidylate synthase ThyX to synthesize thymidylate, which produces H₄PteGlu during the process. Subsequently, data of in vitro enzymatic activity experiments showed that H₄PteGlu hinders PAS activation by competing with hydroxy dihydropteroate (H₂PtePAS) for FolC catalysis. Meanwhile, over-expressing <i>folC</i> in Δ<i>thyA</i> strain and a PAS resistant clinical isolate with known <i>thyA</i> mutation partially restored PAS sensitivity, which relieved the competition between H₄PteGlu and H₂PtePAS. Thus, loss of function mutations in <i>thyA</i> led to increased H₄PteGlu content in bacterial cells, which competed with H₂PtePAS for catalysis by FolC and hence hindered the activation of PAS, leading to decreased production of hydroxyl dihydrofolate (H₂PtePAS-Glu) and finally caused PAS resistance. On the other hand, functional deficiency of <i>thyA</i> in <i>M. tuberculosis</i> pushes the bacterium switch to an unidentified dihydrofolate reductase for H₄PteGlu biosynthesis, which might also contribute to the PAS resistance phenotype. Our study revealed how <i>thyA</i> mutations confer PAS resistance in <i>M. tuberculosis</i> and provided new insights into studies on the folate metabolism of the bacterium.