Toll-like receptor 9 (TLR9) agonists have demonstrated substantial potential as vaccine adjuvants, and as mono- or combination therapies for the treatment of cancer and infectious and allergic diseases. Commonly referred to as CpG oligodeoxynucleotides (ODN), TLR9 agonists directly induce the 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 as vaccine adjuvants, where they can enhance both the humoral and cellular responses to diverse antigens. In mouse tumor models 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. CpG ODN have shown benefit in multiple rodent and primate models of asthma and other allergic diseases, with encouraging results in some early human clinical trials. Although their potential clinical contributions are enormous, the safety and efficacy of these TLR9 agonists in humans remain to be determined.