Although still early in clinical development, agonists of Toll-like receptor 9 (TLR9) have demonstrated potential for the treatment of cancer. TLR9 agonists directly induce activation and maturation of plasmacytoid dendritic cells and enhance differentiation of B cells into antibody-secreting plasma cells. Preclinical and early clinical data support the use of TLR9 agonists in patients with solid tumors and hematologic malignancies. In preclinical studies, TLR9 agonists have shown activity not only as monotherapy, but also in combination with multiple other therapies, including vaccines, antibodies, cellular therapies, other immunotherapies, antiangiogenic agents, radiotherapy, cryotherapy and some chemotherapies. Phase I and II clinical trials have indicated that these agents have antitumor activity as single agents and enhance the development of antitumor T-cell responses when used as therapeutic vaccine adjuvants. The activity and safety of these novel anticancer agents are being explored in a wide range of tumor types as part of a variety of therapeutic strategies with the goal of harnessing the immune response to fight cancer.