Abstract Stimulator of Interferon (IFN) Genes (STING), a cytosolic DNA sensor that recognises cyclic dinucleotides (CDNs), has been an adjuvant target in many cancer immunotherapies. Activation of STING results in the production of Type I IFN through the phosphorylation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) and it is thought this enhances cross-presentation of tumour antigens by dendritic cells (DCs). However, DCs interrogated in many of these studies use in vitro-generated DCs instead of putative ex vivo DCs, and the direct effects of STING activation on different DC subsets are not completely understood. Here, we report that mouse and humanised mouse splenic DC subsets as well as human blood DCs are activated by CDN stimulation and produce Type III IFN (IFN-lambda) but only type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs) produce Type I IFN in response to CDN stimulation. However, only mouse pDCs aberrantly express extremely high levels of CD86 and CD80 and are ablated rapidly after STING activation. Some DC death was also observed in mouse and human cDC2s, but not in human pDCs. This ablation is STING-dependent and occurs via a cell-intrinsic mechanism involving intrinsic apoptosis. These observations demonstrate the differential effects STING activation has on DC subsets as well as highlight a discordance amongst mouse and human DCs during activation, which serve as an important consideration in the translation of animal cancer models and the use of STING ligands as adjuvants in cancer immunotherapy.