Dormancy of Mycobacterium tuberculosis is likely to be a major cause of extended chemotherapeutic regimens and wide prevalence of tuberculosis. The molecular mechanisms underlying M. tuberculosis dormancy are not well understood. In this study, single-copy genes responsible for synthesis (dgc) and degradation (pde) of the ubiquitous bacterial second messenger, cyclic di-GMP (c-di-GMP), were deleted in the virulent M. tuberculosis strain H37Rv to generate dgc(mut) and Δpde, respectively. Under aerobic growth conditions, the two mutants and wild-type cells showed similar phenotypes. However, dgc(mut) and Δpde exhibited increased and reduced dormancy, respectively, in both anaerobiosis-triggered and vitamin C-triggered in vitro dormancy models, as determined by survival and growth recovery from dormancy. The transcriptomes of aerobic cultures of dgc(mut) and wild-type H37Rv exhibited no difference, whereas those of anaerobic cultures showed a significant difference with 61 genes that are not a part of the dosR regulon. Furthermore, Δpde but not dgc(mut) showed decreased infectivity with human THP-1 cells. Δpde also showed attenuated pathogenicity in a C57BL/6 mouse infection model. These findings are explained by c-di-GMP-mediated signaling negatively regulating M. tuberculosis dormancy and pathogenicity.