ARTICLE ABSTRACTCancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)–based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 μmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3–3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 μmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP–killed 4T1 tumor cells, and through c-di-GMP–activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy. Cancer Immunol Res; 2(9); 901–10. ©2014 AACR.