Abstract Plasmacytoid dendritic cells (pDCs) are potent producers of type I and type III IFNs and play a major role in antiviral immunity and autoimmune disorders. The innate sensing of nucleic acids remains the major initiating factor for IFN production by pDCs. TLR-mediated sensing of nucleic acids via endosomal pathways has been studied and documented in detail, whereas the sensing of DNA in cytosolic compartment in human pDCs remains relatively unexplored. We now demonstrate the existence and functionality of the components of cytosolic DNA-sensing pathway comprising cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of IFN gene (STING) in human pDCs. cGAS was initially located in the cytosolic compartment of pDCs and time-dependently colocalized with non-CpG double-stranded immunostimulatory DNA (ISD). Following the colocalization of ISD with cGAS, the downstream pathway was triggered as STING disassociated from its location at the endoplasmic reticulum. Upon direct stimulation of pDCs by STING agonist 2′3′ cGAMP or dsDNA, pDC-s produced type I, and type III IFN. Moreover, we documented that cGAS–STING–mediated IFN production is mediated by nuclear translocation of IRF3 whereas TLR9-mediated activation occurs through IRF7. Our data also indicate that pDC prestimulation of cGAS–STING dampened the TLR9-mediated IFN production. Furthermore, triggering of cGAS–STING induced expression of SOCS1 and SOCS3 in pDCs, indicating a possible autoinhibitory loop that impedes IFN production by pDCs. Thus, our study indicates that the cGAS–STING pathway exists in parallel to the TLR9-mediated DNA recognition in human pDCs with cross-talk between these two pathways.