Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. The pathogenesis by the causative agent,Mycobacterium tuberculosis, is still not fully understood. We have previously reported thatM. tuberculosisRv3586 (disA) encodes a diadenylate cyclase, which converts ATP to cyclic di-AMP (c-di-AMP). In this study, we demonstrated that a protein encoded by Rv2837c (cnpB) possesses c-di-AMP phosphodiesterase activity and cleaves c-di-AMP exclusively to AMP. Our results showed that inM. tuberculosis, deletion ofdisAabolished bacterial c-di-AMP production, whereas deletion ofcnpBsignificantly enhanced the bacterial c-di-AMP accumulation and secretion. The c-di-AMP levels in both mutants could be corrected by expressing the respective gene. We also found that macrophages infected with ΔcnpBsecreted much higher levels of IFN-β than those infected with the wild type (WT) or the complemented mutant. Interestingly, mice infected withM. tuberculosisΔcnpBdisplayed significantly reduced inflammation, less bacterial burden in the lungs and spleens, and extended survival compared with those infected with the WT or the complemented mutant. These results indicate that deletion ofcnpBresults in attenuated virulence, which is correlated with elevated c-di-AMP levels.