In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (RelMtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. RelMtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. RelMtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of RelMtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated RelMtb point mutants that were either synthetase dead (RelMtbH344Y) or hydrolase dead (RelMtbH80A). M. tuberculosis strains expressing the synthetase-dead RelMtbH344Y mutant did not persist in mice, demonstrating that the RelMtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that RelMtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead RelMtb mutant, RelMtbH80A, that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, RelMtbH80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for RelMtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.