Cyclic-di-nucleotide based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although, c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous B. subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Further, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of T. maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue. This article is protected by copyright. All rights reserved.