Because of the emergence of strains of Mycobacterium tuberculosis resistant to first-line antituberculosis agents, one of the second-line drugs, p-aminosalicylate (PAS), has regained importance in the treatment of tuberculosis. The mode of action of PAS, however, remains controversial as to whether it inhibits mycobactin or folate biosynthesis. To unravel this, we have studied the effect of PAS on wild-type Mycobacterium smegmatis and its mutants (gene knockouts of the salicylate pathway –trpE2, entC and entD). The wild type had no sensitivity to PAS (MIC>400 μg mL−1), whereas the mutants were hypersensitive, with 1 μg mL−1 inhibiting growth. The sulphonamides, trimethoprim and dapsone, had little effect on the growth of either the mutants or the wild type. In addition, PAS at 0.5 μg mL−1 increased the accumulation of salicylate with the wild type and mutants. These results support our hypothesis that PAS targets the conversion of salicylate to mycobactin, thus preventing iron acquisition from the host.