Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) have been reported to induce antimycobacterial activity both in vitro and in vivo. The present study analyzes the signals leading to CpG ODN-induced antimicrobial activity in monocytes. In this context, CpG, but not GpC, ODN induced cytosolic Ca²⁺ influx of extracellular origin which, in turn, activated host phospholipase D (PLD). The production of CpG-induced PLD-dependent phosphatidic acid induced the maturation of phagolysosomes and intracellular mycobacterial growth inhibition. These results show the presence of an antimicrobial pathway in monocytes, mediated by Ca²⁺-dependent PLD which can be useful for the exploitation of novel anti-tuberculosis immunotherapy approaches.