Mycobacterium tuberculosis (Mtb) encodes an exceptionally large number of toxin-antitoxin (TA) systems, supporting the hypothesis that TA systems are involved in pathogenesis. We characterized the putative Mtb Rv1044-Rv1045 TA locus structurally and functionally, demonstrating that it constitutes a bona fide TA system but adopts a previously unobserved antitoxicity mechanism involving phosphorylation of the toxin. While Rv1045 encodes the guanylyltransferase TglT functioning as a toxin, Rv1044 encodes the novel atypical serine protein kinase TakA, which specifically phosphorylates the cognate toxin at residue S78, thereby neutralizing its toxicity. In contrast to previous predictions, we found that Rv1044-Rv1045 does not belong to the type IV TA family because TglT and TakA interact with each other as substrate and kinase, suggesting an unusual type of TA system. Protein homology analysis suggests that other COG5340-DUF1814 protein pairs, two highly associated but uncharacterized protein families widespread in prokaryotes, might share this unusual antitoxicity mechanism.