Toxin-antitoxin (TA) systems, stress-responsive genetic elements ubiquitous in microbial genomes, are unusually abundant in the major human pathogen Mycobacterium tuberculosis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown. To address these questions, we have taken a comprehensive approach to identify and functionally characterize all the TA systems encoded in the M. tuberculosis genome. Here we show that 88 putative TA system candidates are present in M. tuberculosis, considerably more than previously thought. Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC), but largely absent from other mycobacteria, including close relatives of M. tuberculosis. We found that many of the M. tuberculosis TA systems are located within discernable genomic islands and were thus likely acquired recently via horizontal gene transfer. We discovered a novel TA system located in the core genome that is conserved across the genus, suggesting that it may fulfill a role common to all mycobacteria. By expressing each of the putative TA systems in M. smegmatis, we demonstrate that 30 encode a functional toxin and its cognate antitoxin. We show that the toxins of the largest family of TA systems, VapBC, act by inhibiting translation via mRNA cleavage. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. The expansion and maintenance of TA genes in the MTBC, coupled with the finding that a subset is transcriptionally activated by stress, suggests that TA systems are important for M. tuberculosis pathogenesis.