Tetracyclines have been one of the most successful classes of antibiotics. However, its extensive use has led to the emergence of widespread drug resistance, resulting in discontinuation of use against several bacterial infections. Prominent resistance mechanisms include drug efflux and the use of ribosome protection proteins. Infrequently, tetracyclines can be inactivated by the TetX class of enzymes, also referred to as tetracycline destructases. Low levels of tolerance to tetracycline in and have been previously attributed to the WhiB7-dependent TetV/Tap efflux pump. However, is ∼500-fold more resistant to tetracycline than and In this report, we show that this high level of resistance to tetracycline and doxycycline in is conferred by a WhiB7-independent tetracycline-inactivating monooxygenase, MabTetX (MAB_1496c). The presence of sublethal doses of tetracycline and doxycycline results in a >200-fold induction of MabTetX, and an isogenic deletion strain is highly sensitive to both antibiotics. Further, purified MabTetX can rapidly monooxygenate both antibiotics. We also demonstrate that expression of MabTetX is repressed by MabTetR, by binding to an inverted repeat sequence upstream of MabTetR; the presence of either antibiotic relieves this repression. Moreover, anhydrotetracycline (ATc) can effectively inhibit MabTetX activity and decreases the MICs of both tetracycline and doxycycline Finally, we show that tigecycline, a glycylcycline tetracycline, not only is a poor substrate of MabTetX but also is incapable of inducing the expression of MabTetX. This is therefore the first demonstration of a tetracycline-inactivating enzyme in mycobacteria. It (i) elucidates the mechanism of tetracycline resistance in , (ii) demonstrates the use of an inhibitor that can potentially reclaim the use of tetracycline and doxycycline, and (iii) identifies two sequential bottlenecks-MabTetX and MabTetR-for acquiring resistance to tigecycline, thereby reiterating its use against .