Toll-like receptor 9 (TLR9), a naturally existing immune regulatory site, not only takes part in enhancing anti-tumor immunity but also promoting the immunosuppressive environment of the growing tumor. TLR9 has shown its potential activity as a therapeutic target in cancer immunotherapy. However, the increased pro-tumor inflammation mediated by TLR9 should also be considered. Therefore, it is important to develop a better understanding of its regulatory mechanisms to improve the therapeutic e?cacy. This review will discuss the mechanisms of immune homeostasis regulated by TLR9 signaling pathways, and also introduce the strategies of how to harness the immune regulatory balance by TLR9 agonists, CpG oligodeoxynucleotides, in cancer immunotherapies.